Treating blood or blood products with compounds which have a mustard, aziridinium or aziridine group and a nucleic acid binding group

ABSTRACT

Methods and compositions for treating pathogens in material are described, including methods of decontaminating human fluids prior to processing in the clinical laboratory and methods for decontaminating blood products prior to in vivo use. The techniques handle large volumes of human serum without impairing the testing results. Novel compounds for photodecontaminating biological material are also contemplated which are compatible with clinical testing, in that they do not interfere with serum analytes.

[0001] This application is a continuation in part of U.S. patentapplication Ser. No. 08/338,040, filed Nov. 14, 1994.

FIELD OF THE INVENTION

[0002] The invention generally relates to new compounds and methods forthe in vitro inactivation of pathogens in biological material intendedfor in vitro or in vivo use, and in particular the inactivation ofpathogens in solutions containing red blood cells, prior to clinicaltesting or transfusion.

BACKGROUND

[0003] The presence of pathogens in blood products, as well as otherbiological materials, is recognized as a significant health problem tohealth workers as well as recipients of the materials.

[0004] With regard to health workers, a great volume of human fluids ishandled daily as part of the routine monitoring of hospital patients byobtaining and testing human fluids (blood, urine, spinal fluid, etc.).Typically, each admitted patient has at least a tube of blood collectedevery day by a phlebotomist. During the transferring, portioning andtesting process, each sample tube is handled by at a clinical workerwhile its contents are exposed. This intensive handling of potentiallyinfectious human fluids is not without health risk. The OccupationalSafety and Health Administration (OSHA) estimates that over five millionhealth workers, including hospital laboratory workers, are exposed toblood borne-pathogen infections in the work place annually. The pathogenresponsible for the overwhelming majority of infections is the hepatitisB virus (HBV). The Center for Disease Control (CDC) estimates there aretwelve thousand cases of HBV infection among health workers each year.Of these cases, over five hundred require hospitalization andapproximately two hundred and fifty of these patients die (i.e. fromfulminant hepatitis, cirrhosis or liver cancer). See Guidelines forPrevention of Transmission of HIV and HBV to Health-Care and PublicSafety Workers, CDC (February 1989). Most full time laboratory employeescontract hepatitis at least once during their career. Indeed, up to onethird of all health care workers show serological evidence of a previousHBV infection. Id.

[0005] Following the recognition of Acquired Immunodeficiency Syndrome(AIDS), clinical laboratories have instituted additional precautions.For example, rather than using manually positioned plastic inserts tomaintain the separation of cells from serum after samples arecentrifuged, a “gel” is now available that is in the empty tube at thetime the blood is drawn. When the tube is centrifuged the cells go belowthe gel while the serum remains above. While the separation can bemaintained in this manner without as much sample handling, this does notreduce the handling of the technologist at the point of analysis.Unfortunately, infectious virus can persist in a liquid or dried statefor prolonged periods of time, possibly even at elevated temperatures.Resnick et al., JAMA 255:1887 (1986).

[0006] Preventative measures such as gloves and eye-wear are notcomplete solutions to the problem. Accidents in the laboratory or clinictypically involve exposure over a larger portion of the body and diseasecan be transmitted through the skin and mucous membranes. Morbidity andMortality Weekly Report 36:285 (1987).

[0007] Clearly, there remains a need for a more adequate solution toblood borne-pathogen infections in the work place. Such a solutionshould serve as a protection against a wide range of pathogens.Furthermore, the mechanics of the solution should not unduly interferewith operations of a laboratory or blood bank.

[0008] Another significant problem is the contamination of the bloodsupply for in vivo use. The safety of the blood supply continues to bethreatened by the transmission of pathogens by transfusion. While thethreat posed by the human immunodeficiency virus (HIV) and the AcquiredImmune Deficiency Syndrome (AIDS) is now widely publicized,contamination of blood products with a number of other blood-borneinfectious viral agents is of even greater concern. See R. Y. Dodd, In:Transfusion Medicine in the 1990's (American Assoc. Blood Banks 1990)(S. J. Nance, ed.). For example, in the United States, it is estimatedthat up to ten (10) percent of multiply transfused recipients develophepatitis accounting for many thousands of cases annually.

[0009] Whole blood collected from volunteer donors for transfusionrecipients is typically separated into its components: red blood cells,platelets, and plasma. Each of these fractions are individually storedand used to treat a multiplicity of specific conditions and diseasestates.

[0010] The red blood cell component is used primarily to treat trauma,chronic anemia, and blood loss due to surgery (particularly cardiac andliver surgery), including postoperative bleeding. D. M. Surgenor et al.Transfusion 32:458 (1992). Approximately twelve (12) million units ofred cells are transfused into approximately four (4) million recipientsannually in the United States alone. E. L. Wallace et al. Transfusion33:139 (1993).

[0011] The safety of the blood supply cannot be assured by merelytesting the blood for pathogens before transfusion. Most testing relieson the detection of antibodies to the pathogen in the prospective blooddonor. It is now well-documented that infectious agents can betransmitted by “seronegative” blood donors, i.e. donors that have nodetectable antibodies to the pathogen. For example, thirteen cases oftransfusion-related AIDS have been reported to the Centers for DiseaseControl (CDC) among recipients of blood that was pre-tested and foundnegative for antibody to the HIV-1 virus.

[0012] Clerical errors and other mistakes further expose patients tocontaminated, incorrectly tested or mislabeled blood. To complicate theproblem, one bad unit can create several victims, since whole blood isroutinely split into components. Mistakes are not infrequent in bloodbanks. Since the beginning of 1990, 29,586 blood bank errors andaccidents have been reported to the FDA. “How Safe Is Our Blood,” U.S.News and World Report, Jun. 27, 1994, 68-78. Recalls by blood centers ofblood released in error are generally ineffective because they takeplace months or years after the blood products have been transfused.

[0013] An alternative approach to eliminate transmission of diseasesthrough blood products is to develop a means to inactivate pathogens intransfusion products. Some of these techniques such as heat [J.Hilfenhous et al. J. Biol. Std. 70:589 (1987)], solvent/detergenttreatment [B. Horowitz et al. Transfusion 25:516 (1985)],gama-irradiation [G. Moroff et al. Transfusion 26:453 (1986)] or UValone [K. N. Proudouz et al. Blood 70:589 (1987)] are completelyincompatible with maintenance of red cell function.

[0014] Another means to inactivate pathogens is the use of methyleneblue. S. J. Wagner et al. examined methylene blue as a virucidal for redcell solutions. S. J. Wagner et al. Transfusion 33:30 (1993). Phototreatment of red cells with methylene blue was found to cause loss ofATP, enhanced ion permeability, and binding of autologous immunoglobulin(IgG) to the red cell surface. It was speculated that some general (andundesirable) modification of the red cell membrane occurs as a result ofthe treatment.

[0015] Yet another approach is to deplete the red cell product ofcontaminating lymphocytes which may harbor viral pathogens. Bothleukodepletion with filters and freeze/thaw procedures have beenexamined. S. M. Bruisten et al. Transfusion 30:833 (1990). Completeremoval of lymphocytes, however, cannot be achieved with such methods.Furthermore, leukodepletion does not address cell-free virus. Thus, thisapproach is not sufficient to render blood completely safe.

[0016] Finally, there is the approach of avoiding blood and using bloodsubstitutes. Hemoglobin solutions, perfluorocarbon emulsions andvesicle-encapsulated hemoglobin have all been suggested as candidates.Unfortunately, each of these has been shown to be inadequate as ageneral substitute. See R. M. Winslow In: Blood Safety: CurrentChallenges (S. J. Nance ed.) (AABB 1992) (pp. 151-167).

[0017] In sum, there is a need for a means of inactivating viralpathogens in red blood cell solutions. This approach must be effectivewithout causing harm to the blood product or the transfusion recipient.

SUMMARY OF THE INVENTION

[0018] The present invention generally relates to new compounds andmethods for the in vitro inactivation of pathogens in biologicalmaterial intended for in vitro or in vivo use, and in particular theinactivation of pathogens in solutions containing red blood cells, priorto clinical testing or transfusion. In accordance with the presentinvention, a compound having a nucleic acid binding ligand and a mustardgroup is selectively employed to treat contamination by nucleicacid-containing microorganisms, including pathogenic viruses. Withoutintending to suggest a mechanism for the present invention, suchcompounds are alkylating agents.

[0019] The present invention contemplates a method of decontaminatingpathogens in a blood product, comprising: adding a compound having amustard group and a nucleic acid binding ligand comprising a psoralengroup or an acridine group to a biological composition suspected ofcontaining pathogens, to create a mixture, said compound reaching afinal concentration sufficient to inactivate substantially all of saidpathogens, and incubating said mixture without significant damage tosaid biological composition. In one embodiment, the compound is added tothe biological composition to a final concentration of said compound ofbetween 1 μg/ml and 250 μg/ml. In another embodiment, the mixture isincubated for between 1 minute and 48 hours. In an embodiment of thepresent invention, the compound is added to the biological composition,the compound is in a mixture comprising dextrose, sodium chloride,mannitol, adenine and H₂O.

[0020] The present invention contemplates that the biologicalcomposition comprises a blood product. The present invention alsocontemplates an additional step: c) transfusing said blood product intoa mammal. In one embodiment, the blood product transfused comprises redblood cells, or specifically, red blood cell concentrate.

[0021] The present invention contemplates the inactivation of both viraland bacterial pathogens. Compounds contemplated by the present inventionfor this method are 8-[3-(Bis-2-chloroethyl)amino]propyloxypsoralen,5-[3-(Bis-2-chloroethyl)aminopropyloxy]methyl-8-methoxypsoralen,5-[3-(Bis-2-chloroethyl)aminopropyloxy]methyl-8-methoxypsoralen, and4′-[4-(Bis-2- chloroethyl)aminobutoxy]methyl-4,5′,8-trimethylpsoralen.The invention contemplates that more than one of said compounds may beadded to the biological composition. Further, the invention contemplatesa step c): after incubating said mixture, removing said compound fromsaid biological composition with an adsorbent material.

[0022] Specifically, the present invention contemplates a method ofinactivating pathogens in a blood product, comprising: a) adding acompound having a mustard group and a nucleic acid binding ligandcomprising a psoralen group or an acridine group to a blood productcomprising red blood cells suspected of containing pathogens, to createa mixture, said compound reaching a final concentration sufficient toinactivate substantially all of said pathogens, and b) incubating saidmixture for between 1 minute and 48 hours, without significant damage tosaid red blood cells. The compound may be added to the blood product toa final concentration of a compound having a nucleic acid binding ligandand a mustard group of between 1 μg/ml and 250 μg/ml. As an additionalcomponent, the invention contemplates that when the compound having anucleic acid binding ligand and a mustard group is added to the bloodproduct, the compound is in a mixture comprising dextrose, sodiumchloride, mannitol, adenine and H₂O. In another embodiment, the methodfurther comprises: c) transfusing said blood product into a mammal. Thepresent invention contemplates the inactivation by this method of bothviral and bacterial pathogens. Further contemplated is a blood productdecontaminated by this method. Various compounds are contemplated foruse in this method, including:8-[3-(Bis-2-chloroethyl)amino]propyloxypsoralen,5-[3-(Bis-2-chloroethyl)aminopropyloxy]methyl-8-methoxypsoralen,5-[3-(Bis-2-chloroethyl)aminopropyloxy]methyl-8-methoxypsoralen, and4′-[4-(Bis-2-chloroethyl)aminobutoxy]methyl-4,5′,8-trimethylpsoralen.Further, more than one of said compounds may be added to said bloodproduct. The present invention contemplates the additional stepcomprising: c) after incubating said mixture, removing said compoundfrom said biological composition with an adsorbent material.

[0023] In yet another embodiment, the present invention contemplatescompositions of matter, comprising:8-[3-(Bis-2-chloroethyl)amino]propyloxypsoralen,5-[3-(Bis-2-chloroethyl)aminopropyloxy]methyl-8-methoxypsoralen,5-[3-(Bis-2-chloroethyl)aminopropyloxy]methyl-8-methoxypsoralen, and4′-[4-(Bis-2-chloroethyl)aminobutoxy]methyl-4,5′,8-trimethylpsoralen.

DESCRIPTION OF THE FIGURES

[0024]FIG. 1 is a graph showing reduction in titer of R17 treated withvarying concentrations of quinacrine mustard in either Adsol or dimethylsulphoxide (DMSO). The horizontal dotted line represents the limit ofdetection of the assay used.

[0025]FIG. 2 is a graph showing inactivation of R17 by quinicrinemustard as a function of hematocrit.

[0026]FIG. 3 is a graph showing the inactivation kinetics of quinicrinemustard.

[0027]FIG. 4 is a graph showing the reduction in titer of R17 as afunction of time of incubation of quinicrine mustard in Adsol.

[0028]FIG. 5 is a graph showing the reduction in R17 inactivationactivity as a function of time when incubated in the presence of eitherAdsol, red blood cells or Amberlite XAD-16™.

[0029]FIG. 6 is a graph showing the effects of quinicrine mustard atvarying concentrations on extra-cellular potassium levels.

[0030]FIG. 7 is a graph showing the reduction in titer of R17 treatedwith varying concentrations of quinicrine mustard; the horizontal dottedline represents the limit of detection of the assay used.

[0031]FIG. 8 is a graph showing the activity of quinicrine mustard,after incubation in red blood cells, with or without the presence ofAmberlite XAD-16™, in an Ames assay using strain TA 1537.

[0032]FIG. 9 is a graph showing the inactivation of a bacterial strain,Staphylococcus Epidermis, using quinicrine mustard at varyingconcentrations.

DESCRIPTION OF THE INVENTION

[0033] The present invention generally relates to new compounds andmethods for the in vitro inactivation of pathogens in biologicalmaterial intended for in vitro or in vivo use, and in particular theinactivation of pathogens in solutions containing red blood cells, priorto clinical testing or transfusion. In accordance with the presentinvention, a compound having a nucleic acid binding ligand and a mustardgroup is selectively employed to treat contamination by nucleicacid-containing microorganisms, including pathogenic viruses andbacteria.

[0034] I. Compounds of the Present Invention

[0035] Red blood cell decontamination methods using photoactivatedcompounds have in the past encountered a problem due to the absorbency,by hemoglobin, of light at wavelengths necessary to activate compounds.Thus, even though the previous methods would inactivate pathogens inother media, they are inefficient in the presence of red blood cells. Incontrast, the present invention contemplates a method of sterilizationcapable of effectively inactivating pathogens even in red cellconcentrates [hematocrits ranging from 1% to 60% or higher].

[0036] The present invention contemplates treating red blood cellsolutions with a compound which inactivates pathogens without requiringexposure to light. The advantage of the present invention forinactivation in fluids for transfusion is two-fold. First, light is notrequired, allowing for a less complex technology for inactivation.Second, the decontamination compound is completely reacted after a shortamount of time. The treatment is complete in several minutes or hours,depending on the compound used. Material which does not react withnucleic acids or another biomolecule hydrolyzes, leaving no compound tobe transfused.

[0037] Without intending to be limited to any particular mechanism ofaction of the present invention, compounds of the present invention havetwo characteristics in common. The first characteristic is that theybind nucleic acid non-covalently. The second is that they have at leastone mustard group.

[0038] A. Non-covalent Nucleic Acid Binding Group

[0039] A compound which binds nucleic acid has a “nucleic acid bindingligand”, herein defined as a group which has an affinity for and canbind to nucleic acids non-covalently. There are several modes of bindingto nucleic acids. Compounds which bind by any of the following modes,combinations of them, or other modes are considered nucleic acid bindingligands. While the invention is not limited to the following compounds,some examples of nucleic acid binding ligands are: a) intercalators,such as acridines, acridones, proflavin, acriflavine, actinomycins,anthracyclinones, beta-rhodomycin A, daunamycin, thiaxanthenones,miracil D, anthramycin, mitomycin, echinomycin, quinomycin, triostin,diacridines, ellipticene (including dimers, trimers and analogs),norphilin A, fluorenes and flourenones, fluorenodiamines, quinacrine,benzacridines, phenazines, phenanthradines, phenothiazines,chlorpromazine, phenoxazines, benzothiazoles, xanthenes andthio-xanthenes, anthraquinones, anthrapyrazoles, benzothiopyranoindoles,3,4-benzpyrene, benzopyrene diol epoxidie, 1-pyrenyloxirane,benzanthracene-5,6-oxide, benzodipyrones, benzothiazoles, quinolones,chloroquine, quinine, phenylquinoline carboxamides, furocoumarins, suchas psoralens and isopsoralens, ethidium salts, propidium, coralyne,ellipticine cation and derivatives, polycyclic hydrocarbons and theiroxirane derivatives, and echinimycin; b) minor groove binders such asdistamycin, mitomycin, netropsin, other lexitropsins, Hoechst 33258 andother Hoechst dyes, DAPI (4′,6′-diamidine-2-phenylindole), berenil, andtriarylmethane dyes; c) major groove binders such as aflatoxins; d)molecules that bind by electrostatics (phosphate backbone binders), suchas spermine, spermidine, and other polyamines; e) nucleic acids oranalogues which bind by such sequence specific interactions as triplehelix formation, D-loop formation, and direct base pairing to singlestranded targets.

[0040] While not limited to any particular mechanism, it is believedthat the nucleic acid binding ligand functions as a carrier (or anchor)that targets (or directs) the molecule to nucleic acid, interactingnon-covalently therewith.

[0041] 1. Psoralens as Noncovalent Nucleic Acid Binding Groups

[0042] The present invention contemplates a specific class of compoundswhich use a psoralen group as a nucleic acid binding group. Thesecompounds are particularly suitable for use in the present invention.Previous nucleic acid specific alkylating agents typically contain analkylating moiety such as a chloroethylamine fragment, connected to anucleic acid specific group, an intercalator (e.g., acridine), or aminor groove binder. These moieties are mutagenic in themselves. Afterthe residual alkyating agent has been hydrolysed from the compound, theresidue may still be rather mutagenic. In contrast, compounds having apsoralen nucleic acid binding group display substantially reducedmutagenicity, thus providing an improved safety factor. Psoralens arewell known as nucleic acid intercalators but their utility has mainlybeen as photoactive agents which covalently bind to the nucleic acidsupon irradiation with UVA (320-400 nm). Without intending to be bound toany mechanism of action of the present invention, it is hypothesizedthat the role of the psoralen group in the compounds described here isas an intercalator to increase the specificity of location of thealkylating agent, and thus the specificity of the alkylation reaction tonucleic acids.

[0043] Specifically some novel compounds of the present inventioncontain psoralens [7H-furo(3,2-g)-(1)-benzopyran-7-one, or b-lactone of6-hydroxy-5-benzofuranacrylic acid], which are linear molecules:

[0044] and in which the two oxygen residues appended to the centralaromatic moiety have a 1, 3 orientation, and further in which the furanring moiety is linked to the 6 position of the two ring coumarin system.Psoralen derivatives are derived from substitution of the linearfurocoumarin at the 3, 4, 5, 8, 4′, or 5′ positions.

[0045] A psoralen of the present invention is represented in thestructure below, wherein one or two of R₁, R₂, R₃, R₄, or R₅ are a(2-chloroethyl)amino group, optionally with a second 2-chloroethyl groupon the amine, attached to a psoralen by a chain of one to nine carbons.The chain can contain one or more heteroatoms of the group comprisingoxygen, nitrogen or sulfur. The chain can optionally contain one or moreunsaturated bonds or carbonyl groups. The chain is optionallysubstituted with lower alkyl groups.

[0046] Specifically the novel compounds contain a psoralen where one ortwo of R1, R2, R3, R4, or R5 are a (2-chloroethyl)amino group or(2-bromoethyl)amino group, optionally with a second 2-haloethyl group onthe amine, attached to a psoralen by a chain of one to nine carbons. Thechain can contain one or more heteroatoms of the group comprisingoxygen, nitrogen or sulfur. The chain can optionally contain one or moreunsaturated bonds or carbonyl groups. The chain is optionallysubstituted with lower alkyl groups.

[0047] Positions R1-R5 unoccupied by the alkylating group may behydrogen, lower alkyl, lower alkoxy, halogen, CH2OR6 or CH2NR7R8 (whereR6-R8 are hydrogen or lower alkyl).

[0048] The compounds may be neutral amines, or their salts.

[0049] Ring construction of psoralens, and their functionalization withdisplaceable groups, X, (where X=Cl, Br, I, OSO₂CH₃, etc,) is describedin the literature (Hearst et al, U.S. Pat. Nos. 4,124,598; 4,196,281;Kaufman, U.S. Pat. Nos. 4,269,851; 4,269,852; 4,294,822; 4,298,614;4,370,344; Wollowitz et al., U.S. Pat. No. 5,399,719; Antonello, S. C.,et al., Farmaco (1978) 34, 139).

[0050] The desired products are constructed by one of three routes. Inthe first, a 2-hydroxyethylamine (e.g., diethanolamine) is reacteddirectly with psoralen-(CH₂)_(n)X where X is a readily displaceablegroup such as a halide, mesylate or tosylate. The chain is attached tothe psoralen at the 3, 4, 4′, 5′, or 8 positions, other substituents maybe on the psoralen ring, and n=1-6. In a second steps, the hydroxygroups of the intermediate are then converted to chloro or bromo groupsby standard means, for example with thionyl chloride to give the desiredproduct.

[0051] In the second route, the functionalized psoralen is reacted withHY—(CH₂)_(m)—OH, where Y=NH, S, O and m=2-6). The terminal alcohol isthen converted to a readily displaceable group (halo, mesylate, etc.) bystandard means, then reacted with the (2-hydroxyethyl)amine. Theresultant compound is converted into a haloethylamine-functionalizedproduct as described above.

[0052] In the final route, the HY—(CH₂)_(m)—N(CH₂CH₂OH)₂, where m=2 to6, is prepared as described in the literature (e.g., Peck, R. M.,Preston, R. K., Creech, H. J., J. Amer. Chem. Soc., (1959) 81, 3984),and reacted directly with the functionalized psoralen. Again, conversionof the hydroxy groups to halides gives the desired psoralen mustardproducts.

[0053] B. Mustard Group

[0054] The second characteristic that compounds of the present inventionhave in common is that they contain at least one mustard group. A“mustard group” is defined here as including mono or bis haloethylaminegroups, and mono haloethylsulfide groups.

[0055] The present invention is not limited strictly to mustards. It isbelieved that mustards can form reactive intermediates such asaziridinium or aziridine complexes and sulfur analogs of thesecomplexes. The present invention also contemplates functional groupsthat are the equivalent of mustards, such as epoxides.

[0056] While not limited to any particular mechanism, compounds havingmustard groups are known to react with nucleic acids to form covalentcomplexes which inhibit nucleic acid replication. They are typicallysolids that, upon dissolution in a medium which contains nucleophiles,completely react within minutes or hours. Some examples are shown below.

[0057] Nitrogen mustards are members of the class of compounds, having anucleic acid binding ligand and a mustard group, which are thoroughlydescribed in the literature. E.g., see Gravatt, G. L., et al.,“DNA-Directed Alkylating Agents. 4. 4-Anilinoquinoline-Based MinorGroove Directed Aniline Mustards,” J. Med. Chem. 34:1552 (1991);Cummings, J., et al., “Determination of Reactive Nitrogen MustardAnticancer Drugs in Plasma by High-Performance Liquid ChromatographyUsing Derivatization,” Anal. Chem. 63:1514 (1991). They are known to bepotent alkylators of nucleic acid and due to this mode of action, theyhave been widely studied as anti-tumor agents. Several have foundpractical use in the clinic (e.g. aniline mustard, chlorambucil,melphalan).

[0058] One class of nitrogen mustards is the aniline mustards. Thesecompound have at least one haloethylaminoaniline group on it, where thehaloethyl may be mono or bis. An example of a bis(haloethyl)aminoanilinegroup appears below (where R is the point of linkage to other groups):

[0059] A specific aniline mustard group is the acridine carried anilinemustards (described in Gravatt, et al., J. Med. Chem. 34:1552), where Rcomprises a linking group (for example O, CH₂, S, COHN, or CO, however,other linking groups are contemplated) which links the mustard group toa second component, an acridine group. An example of the components of a9-aminoacridine carried aniline mustard appears below (where X is thelinking group):

[0060] The present invention demonstrates that a specific compoundhaving a nucleic acid binding ligand and a mustard group,N1,N1-bis(2-chloroethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-1,4-pentanediaminedihydrochloride (“quinicrine mustard”), is useful as an antiviral agentfor red cells. Quinicrine mustard is commercially available (fromAldrich, Milwaukee, Wis., as quinicrine mustard dihydrochloride hydrate,structure shown below).

[0061] II. Materials for Decontamination

[0062] The present invention contemplates novel compounds and a new usefor compounds having a nucleic acid binding ligand and a mustard group:the inactivation of viruses and bacteria in blood, blood products andother biological compositions. While not an exclusive list, thefollowing biological compositions are contemplated, and are referred togenerally as “samples”. Of the blood and blood components contemplated,exemplary compositions include whole blood, packed red cells, platelets,plasma (fresh or fresh frozen plasma), and proteins derived from bloodor blood components. Blood components also encompass plasma proteinportion, antihemophilic factor (AHF, Factor VIII); Factor IX and FactorIX complex (Factors II, VII, IX and X); fibrinogens, Factor XIII,prothrombin and thrombin (Factor II and IIa); immunoglobulins (e.g. IgA,IgD, IgE, IgG and IgM and fragments thereof e.g. Fab, F(ab′)₂, and Fc);hyper-immune globulins as used against tetanus and hepatitis B;cryoprecipitate; albumin; interferons; lymphokines; and transferfactors. The present invention also contemplates, as part of blood andblood products, a synthetic version of any blood or blood product.

[0063] Other biological compositions which are contemplated by thepresent invention include vaccines, recombinant DNA produced proteins,oligopeptide ligands, etc. Biological compositions also encompassclinical samples other than blood and blood components, such as urine,sputum, feces, spinal fluid, and other materials removed from mammalsfor clinical testing.

[0064] III. Inactivation of Pathogens

[0065] The present invention contemplates treating a blood product witha compound having a nucleic acid binding ligand and a mustard group toinactivate contaminating pathogen nucleic acid sequences before usingthe blood product.

[0066] A. Inactivation in General

[0067] The term “inactivation” is here defined as the altering of thenucleic acid of a unit of pathogen so as to render the unit of pathogenincapable of replication. This is distinct from “total inactivation”,where all pathogen units present in a given sample are renderedincapable of replication, or “substantial inactivation,” where most ofthe pathogen units present are rendered incapable of replication.“Inactivation efficiency” of a compound is defined as the level ofinactivation the compound can achieve at a given concentration ofcompound or dose of irradiation. For example, if 100 μM of ahypothetical compound X inactivated 5 logs of HIV virus whereas underthe same experimental conditions, the same concentration of compound Yinactivated only 1 log of virus, then compound X would have a better“inactivation efficiency” than compound Y.

[0068] To appreciate that an “inactivation” method may or may notachieve “total inactivation,” it is useful to consider a specificexample. A bacterial culture is said to be inactivated if an aliquot ofthe culture, when transferred to a fresh culture plate and permitted togrow, is undetectable after a certain time period. A minimal number ofviable bacteria must be applied to the plate for a signal to bedetectable. With the optimum detection method, this minimal number is 1bacterial cell. With a sub optimal detection method, the minimal numberof bacterial cells applied so that a signal is observed may be muchgreater than 1. The detection method determines a “threshold” belowwhich the “inactivation method” appears to be completely effective (andabove which “inactivation” is, in fact, only partially effective).

[0069] B. Inactivation of Potential Pathogens

[0070] The same considerations of detection method and threshold existwhen determining the sensitivity limit of an inactivation method fornucleic acid. Again, “inactivation” means that a unit of pathogen isrendered incapable of replication.

[0071] In the case of inactivation methods for material to be used byhumans, whether in vivo or in vitro, the detection method cantheoretically be taken to be the measurement of the level of infectionwith a disease as a result of exposure to the material. The thresholdbelow which the inactivation method is complete is then taken to be thelevel of inactivation which is sufficient to prevent disease fromoccurring due to contact with the material. It is recognized that inthis practical scenario, it is not essential that the methods of thepresent invention result in “total inactivation”. That is to say,“substantial inactivation” will be adequate as long as the viableportion is insufficient to cause disease. Thus “substantially all” of apathogen is inactivated when any viable portion of the pathogen whichremaining is insufficient to cause disease. The inactivation method ofthe present invention renders nucleic acid in pathogens substantiallyinactivated. In one embodiment, the inactivation method renders pathogennucleic acid in blood preparations substantially inactivated.

[0072] Without intending to be limited to any method by which thecompounds of the present invention inactivate pathogens, it is believedthat inactivation results from alkylation of portions of the pathogennucleic acid. Further, while it is not intended that the inactivationmethod of the present invention be limited by the nature of the nucleicacid; it is contemplated that the inactivation method render all formsof nucleic acid (whether DNA, mRNA, etc.) substantially inactivated.

[0073] When a compound having a nucleic acid binding ligand and amustard group is used to modify nucleic acid, the interaction of thepathogen nucleic acid (whether DNA, mRNA, etc.) with the compoundpreferably prevents replication of the pathogen, such that, if a humanis exposed to the treated pathogen, infection will not result.

[0074] “Synthetic media” is herein defined as an aqueous synthetic bloodor blood product storage media. In one embodiment, the present inventioncontemplates inactivating blood products in synthetic media comprising abuffered saline solution.

[0075] The present method inactivates nucleic acid based pathogenspresent in blood through a single procedure. Thus, it has the potentialto eliminate bacteria, protozoa, and viruses as well. It is not intendedthat the present invention be limited by the number or nature ofpathogens inactivated. Importantly, however, the treatment of thepresent invention has been found to block the replication of the HIVvirus. Had an effective decontamination method been available prior tothe advent of the AIDS pandemic, no transfusion associated HIVtransmission would have occurred. Decontamination based on compoundshaving a nucleic acid binding ligand and a mustard group has thepotential to eliminate all infectious agents from the blood supply,regardless of the pathogen involved.

[0076] C. Selecting Compounds for Inactivation of Pathogens

[0077] In order to evaluate a compound to decide if it would be usefulin the decontamination methods of the present invention, two importantproperties should be considered: 1) the compound's ability to inactivatepathogens and 2) its mutagenicity after treatment. The ability of acompound to inactivate pathogens may be determined by several methods.One technique is to perform a bacteriophage screen; an assay whichdetermines nucleic acid binding of test compounds. A screen of thistype, an R17 screen, is described in detail in an example, below. If theR17 screen shows inactivation activity, it is useful to directly testthe compound's ability to inactivate a virus. One method of performing adirect viral inactivation screen is described in detail in an examplebelow for cell free HIV.

[0078] The R17 bacteriophage screen is believed to be predictive of HIVinactivation efficiency, as well as the efficiency of compounds againstmany other viruses. R17 was chosen because it was expected to be a verydifficult pathogen to inactivate. It is a small, single stranded RNAphage. Without intending to be limited to any means by which the presentinvention operates, it is expected that shorter pieces of nucleic acidare harder to inactivate because they provide a smaller target for thecompound. Thus it is expected that under conditions that result in theinactivation of R17 the inactivation of many viruses and bacteria willalso be obtained.

[0079] The cell free HIV screen complements the R17 screen by affirmingthat a given compound which has tested positive in R17 will actuallywork effectively to inactivate viruses. Thus, if a compound showsactivity in the R17 screen, it is next tested in the viral inactivationscreen.

[0080] The second property that is important in testing a compound foruse in methods of the present invention is mutagenicity after treatment.The most widely used mutagen/carcinogen screening assay is the Amestest. This assay is described by D. M. Maron and B. N. Ames in MutationResearch, 113: 173 (1983) and a specific screen is described in detailin an example, below. The Ames test utilizes several unique strains ofSalmonella typhimurium that are histidine-dependent for growth and thatlack the usual DNA repair enzymes. The frequency of normal mutationsthat render the bacteria independent of histidine (i.e., the frequencyof spontaneous revertants) is low. The test allows one to evaluate theimpact of any residual chemical entities that remain after treatment onthis revertant frequency.

[0081] Because some substances are not mutagenic by themselves, but areconverted to a mutagen by metabolic action, the compound to be tested ismixed with the bacteria on agar plates along with the liver extract. Theliver extract serves to mimic metabolic action in an animal. Controlplates have only the bacteria and the extract.

[0082] The mixtures are allowed to incubate. Growth of bacteria (if any)is checked by counting colonies. A positive Ames test is one where thenumber of colonies on the plates with mixtures containing the compoundsignificantly exceeds the number on the corresponding control plates.

[0083] When known carcinogens are screened in this manner with the Amestest, approximately ninety percent are positive. When knownnoncarcinogens are similarly tested, approximately ninety percent arenegative.

[0084] A compound (X) can be evaluated as a potential decontaminationcompound for use in the present invention, as shown in Table 1, below. Xis initially evaluated in Step I. X is screened in the R17 assay, in thepresence of red blood cells, at several different concentrations between4 and 320 μM, as explained in an example below. If the compound showsinactivation activity greater than 1 log inactivation of R17 (log kill)in the R17 screen at any concentration, the compound is then screened inthe cell free HIV assay, Step II, as explained in an example below. Ifthe compound shows inactivation activity greater than 1 log inactivationof HIV (log kill) in the cell free HIV assay, the compound is a usefulagent for inactivation of pathogens in clinical test samples. If thecompound is being evaluated for decontamination of biological materialsto be used in vivo, it is then taken through Step III. A biologicalmaterial decontaminated by a method of the present invention is screenedin the Ames assay to determine whether any compound that remains afterdecontamination is mutagenic. Finally, if the residual material does notshow significant mutagenicity in the Ames assay, the compound isidentified as a useful agent for inactivation of pathogens in productsto be used in vivo as well. TABLE 1 STEP SCREEN RESULT INTERPRETATION IR17 >1 log kill by any potential compound, concentration go to step 2 <1log kill compound is ineffective as an inactivation treatment II ViralInactivation >1 log kill by any useful for clinical concentration sampledecontamination go to step 3 <1 log kill compound is ineffective as aninactivation treatment III Ames less mutagenic useful agent for than AMTinactivation

[0085] By following these instructions, a person can determine whichcompounds would be appropriate for use in methods of the presentinvention.

[0086] D. Delivery and Removal of Compounds for Inactivation

[0087] The present invention contemplates several different formulationsand routes by which the compounds described herein can be delivered inan inactivation method, and where desired, removed. This section ismerely illustrative, and not intended to limit the invention to any formor method of treatment with the compounds.

[0088] The compounds of the present invention may be introduced in aninactivation method in several forms and at various times, which maydepend on the purpose for which the blood preparation is decontaminated.The compounds may, for example, be introduced as an aqueous solution inwater, saline, a synthetic media or a variety of other media. Thecompounds may alternatively be provided as dry formulations, with orwithout adjuvants. Further, the compounds may be introduced alone, or ina “cocktail” or mixture of several different compounds: In a preferredembodiment, a compound having a nucleic acid binding ligand and amustard group is employed at a concentration less than 250 μM.

[0089] The compounds can be mixed directly with the blood or bloodproduct or prepared as a solution or suspension in a bio-compatiblefluid [such as Adsol (the contents of which are set forth in theExperimental section, below) or an organic solvent (e.g. dimethylsulfoxide (DMSO), ethanol, glycerin, polyethylene glycol (PEG) orpolypropylene glycol)] and then mixed with the blood. The new compoundsmay also be provided at different points in the inactivation process.For example, the compound may be introduced to the reaction vessel, suchas a blood bag, at the point of manufacture. Alternatively, the compoundmay be added to the material to be sterilized after the material hasbeen placed in the reaction vessel.

[0090] 1. Decontamination of Clinical Samples

[0091] A clinical sample is defined as any material removed from mammalsfor clinical testing, including, but not limited to blood and bloodcomponents, urine, sputum, feces, bone marrow, and spinal fluid. A serumanalyte is defined here as a component found in clinical samples whichis measured in clinical chemistry tests. Examples of serum analytesinclude, but are not limited to: glucose, blood urea nitrogen,creatinine, blood urea nitrogen/creatinine ratio, sodium, potassium,chloride, magnesium, calcium, phosphorous inorganic, total protein,albumin, total globulin, albumin/globulin ratio, billirubin, alkalinephosphatase, lactate dehydrogenase, glutamate transferase, aspartatetransaminase, alanine aminotransferase, uric acid, iron, triglycerides,and cholesterol.

[0092] In the decontamination of clinical samples, the goal is todecontaminate the sample so that infectious agents cannot be transferredto clinical laboratory workers. Because the samples will not betransfused into a recipient, there is less concern that residualcompound be removed from the sample. Thus scrubbing techniques may notbe desired. The present invention contemplates that the compound may bein the clinical sample test tube prior to drawing the sample from thepatient, or it may be added after drawing. Once the compound hascontacted the sample, the sample preferably is thoroughly mixed, thenincubated. The sample may then be screened in the desired panel ofclinical chemistry tests without concern for spreading infectiousdiseases.

[0093] 2. Decontamination of Blood Products for Transfusion

[0094] The compound for decontamination may be introduced to the wholeblood prior to fractionating, by adding to the blood bag before or afterblood is drawn. Alternatively, the compound may be added afterfractionation of the blood, decontaminating the individual fractions.

[0095] In products for transfusion, in some cases it may be desirable toremove residual compound or chemical products of the reaction aftertreatment of the product but prior to transfusion. The present inventioncontemplates the removal, or “scrub” of the compound from the bloodproduct post illumination and prior to transfusion. In one embodiment,any residual compound or chemical product may be removed using anadsorbent material. Examples of adsorbent materials which may be used inthe present invention include, but are not limited to: activatedcharcoal (either uncoated or coated with a polymer), silica, reversephase silica, polymeric adsorbents, and modified polymeric adsorbents.The present invention contemplates several ways for the introduction ofthe adsorbent material to the blood products for transfusion. Theadsorbent may be mixed directly with the blood products and subsequentlyfiltered out. Alternatively, the blood products could be passed througha filter containing the adsorbent material.

[0096] 3. Decontamination of Vaccines and Other Biological Compositions

[0097] Vaccines and other biological compositions which are not derivedfrom blood, such as recombinant DNA produced proteins and oligopeptideligands, may also be decontaminated using methods of the presentinvention. Recombinant DNA produced proteins often are manufactured inlarge quantities in host organisms. Introduction of the decontaminationcompound may occur prior to amplification, so that as the host organismsgrow, the compound is incorporated into the organism. Alternatively, thecompound may be added after manufacture, but before the product isintroduced into a mammal.

[0098] Removal of the compound before use may be desired here as well aswith blood products for transfusion. Those methods mentioned above applyequally well in the case of vaccines and other biological compositions.

[0099] V. Preservation of Biochemical Properties of Treated Material

[0100] When treating blood products to be used in vivo, one must askwhether the process or the compounds used alter the in vivo activity ofthe treated material. For example, red blood cell transfusion is a wellestablished efficacious treatment for patients suffering large bloodloss. However, if the inactivation treatment used greatly reduces the invivo life of the red blood cells, then the treatment has no practicalvalue. The compounds of the present invention are useful in inactivationprocedures because the reaction can be carried out at temperaturescompatible with retaining biochemical properties of blood and bloodproducts. But not all methods of pathogen inactivation will inactivatewithout significantly lowering the biological activity of thedecontaminated material. Previously known compounds and protocols forinactivation have necessitated both exposure to light and the subsequentremoval of molecular oxygen from the reaction before and during theexposure, to prevent damage to blood products from oxygen radicalsproduced during irradiation. See L. Lin et al., Blood 74:517 (1989);U.S. Pat. No. 4,727,027, to Wiesehahn. The present invention may be usedto decontaminate blood products without light, and in the presence ofoxygen, without destroying the activity for which the products areprepared. Further, with methods of the present invention, there is noneed to reduce the concentration of molecular oxygen.

[0101] The present invention contemplates that in vivo activity of ablood product is not destroyed or significantly lowered if the bloodproduct which is decontaminated by methods of the present inventiontests as would a normally functioning blood product blood product inknown assays for function of the particular blood product. The activityof a clinical sample is not destroyed or significantly lowered if theclinical sample which is decontaminated by methods of the presentinvention tests as would an untreated sample in common clinicalchemistry tests. In contrast, a blood product or clinical sample isconsidered to have incurred “significant damage” when the blood productno longer functions for the purpose it was prepared. For example, wherered blood cells are concerned, in vivo activity is not destroyed orsignificantly lowered if AMP levels, IgG binding, and extracellularpotassium levels of the red blood cells are substantially the same inred blood cells treated by the methods of the present invention andstored 9 days as they are in untreated samples stored for 9 days.Similarly, a clinical sample has not suffered significant damage if atreated sample tests substantially the same as an untreated sample inone or more common clinical chemistry tests. “Substantially the same”means that the values of the treated samples do not exhibit change whichis more than 10% larger than change in values exhibited in a non treatedcontrol. In the case of red blood cells, this comparison is made after a9 day storage following treatment.

[0102] VI. Preparation of Vaccines

[0103] The preparation of viral vaccines is also contemplated by methodsof the present invention. The present invention contemplates producingvaccines to a wide variety of viruses, including human viruses andanimal viruses, such as canine, feline, bovine, porcine, equine andovine viruses. The contemplated method is suitable for inactivatingdouble stranded DNA viruses, single stranded DNA viruses,double-stranded RNA viruses and single-stranded RNA viruses, includingboth enveloped and non-enveloped viruses. A contemplated method forproducing a vaccine for inoculation of a mammalian host susceptible toinfection by a virus comprises growing culture of virus, isolated froman infected host, in a suitable mammalian cell culture, exposing atleast one of the seed viruses to a compound having a nucleic acidbinding ligand and a mustard group for a time sufficient to inactivatethe virus to a non-infectious degree, under conditions whichsubstantially preserve the antigenic characteristics of the inactivatedviral particles, and combining said inactivated virus with a suitableadjuvant.

[0104] The inactivated virus may be formulated in a variety of ways foruse as a vaccine. The concentration of the virus will generally be fromabout 10⁶ to 10⁹ plaque forming units (pfu)/ml, as determined prior toinactivation, with a total dosage of at least 10⁵ plaque forming unitsper dose (pfu/dose), usually at least 10⁶ pfu/dose, preferably at least10⁷ pfu/dose. The total dosage will usually be at or near about 10⁹pfu/dose, more usually being about 10⁸ pfu/dose. The vaccine may includecells or may be cell-free. It may be an inert physiologically acceptablemedium, such as ionized water, phosphate-buffered saline, saline, or thelike, or may be administered in combination with a physiologicallyacceptable immunologic adjuvant, including but not limited to mineraloils, vegetable oils, mineral salts, and immunopotentiators, such asmuramyl dipeptide. The vaccine may be administered subcutaneously,intramuscularly, intraperitoneally, orally, or nasally. Usually, aspecific dosage at a specific site will range from about 0.1 ml to 4 ml,where the total dosage will range from about 0.5 ml to 8 ml. The numberof injections and their temporal spacing may be highly variable, butusually 1 to 3 injections at 1, 2 or 3 week intervals are effective.

EXPERIMENTAL

[0105] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0106] In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); L (liters);ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters);μm (micrometers); nm (nanometers); ° C. (degrees Centigrade); HPLC (HighPressure Liquid Chromatography); Q (quinacrine); QM (quinacrinemustard); DMSO (dimethylsulfoxide); Htc (hematocrit); RBC (red bloodcell); LB (Luria Broth); N-acetyl-cysteine (NAC); BUN (blood ureanitrogen); Creat. (creatinine); phos acid (phosphoric acid); alk(alkaline phosphatase); ALT (Alanine Aminotransferase); AST (AspartateTransaminase); LDH (lactate dehydrogenase); GGT (Glutamate Transferase);cfu (culture forming units); pfu (plaque forming units); DMEM(Delbecco's modified eagles medium); FBS (fetal bovine serum); PRBC(packed red blood cells); PCR (polymerase chain reaction); rpm(revolutions per minute); TC (tissue culture); NHSP (normal human serumpool); LSM (lymphocyte separation medium); NCS (newborn Calf Serum); PBS(phosphate buffered saline).

[0107] While it is available commercially from Baxter Heathcare Corp,Deerfield, Ill., Adsol used in the following experiments was made bysterile filtering the following mixture: 22 g glucose, 9 g NaCl, 7.5 gmannitol, and 0.27 g adenine in 1 liter of distilled H20.

[0108] The polymerase chain reaction (PCR) is used in some of theexamples below. PCR is a method for increasing the concentration of asegment of a target sequence in a mixture of genomic DNA without cloningor purification. See K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and4,683,202, hereby incorporated by reference. This process for amplifyingthe target sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then areannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing, and polymerase extension can be repeated many times(i.e. denaturation, annealing and extension constitute one “cycle;”there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to by theinventors as the “polymerase chain reaction”.

Example 1

[0109] This example measures the R17 inactivation activity of quinicrinemustard (QM) solutions made in either Adsol or DMSO. The bacteriophageR17 has a single stranded RNA genome of approximately 1.2×10⁶ daltons,and is difficult to inactivate compared to many other targets. Seegenerally L. Lin et al., Blood 74:517 (1989). The advantage of the R17system is that inactivation can be readily assayed in the laboratory.

[0110] The assay used to determine inactivation measures the ability ofthe phage to subsequently infect bacteria and inhibit their growth. Thephage was grown up in Hrf 3000 bacteria. (R17 and Hrf 3000 were obtainedfrom American Tissue Culture Collection (ATCC), Washington, D.C.).First, the R17 stock virus was diluted (10.9 logs/ml in LB broth) 1:20in Adsol (R17-Adsol). Then a 30% hematocrit (Htc) red blood cellconcentrate in R17-Adsol mixture was prepared by spinning down red bloodcells (RBC) from whole blood and resuspending 3.5 ml RBC pellet in 7.0ml R17-Adsol. In this, and the following experiments, Htc was measuredon a Model F800 Sysmex cell counter (Toa Medical Electronics, Kobe,Japan). Ten 1 ml aliquots of the samples were then transferred tosterile tubes.

[0111] Approximately 2 mg of QM, commercially available from Aldrich,Inc., Milwaukee, Wis., was weighed out into each of two tubes. Sampleswere then dissolved in DMSO or Adsol, respectively, to a finalconcentration of 0.4 mg/ml. QM in Adsol is a suspension, not a solution,at this concentration.

[0112] Next, the QM suspension was added to the R17-Adsol samples toachieve the following final concentrations of QM in the sample tubes:2.5, 5.0, 10, or 20 μg/ml. The QM was completely solubilized at theseconcentrations. Positive control samples were also prepared, where 50 μlof either Adsol or DMSO was added to R17-Adsol samples. The samples wereallowed to stand at room temperature for at least 1 hour. Then thesamples were titered by an R17 phage assay. Sterile 13 ml dilution tubeswere prepared with LB broth. To make the dilutions, a 0.1 ml aliquot ofthe solution of phage was added to the first dilution tube of 0.4 ml ofmedia. Then 0.02 ml of this solution was added to the second tube of 0.5ml media (1:25). The second solution was then diluted serially (1:25)into the remaining tubes. To each diluted sample was added 0.05 ml ofHrf 3000 bacteria cultured overnight and 3 ml of molten LB top agar. Themixed materials were poured onto LB broth plates. After the top agarhardened, the plates were incubated at 37° C. overnight. Plaques werecounted the following morning and the titer of the phage remaining aftertreatment was calculated based on the dilution factors.

[0113] The results are shown in Table 2, below, and FIG. 1. It is clearfrom the data that even at concentrations as low at 2.5 μg/ml QM iseffective in inactivating R17. At concentrations above 10 μg/ml,complete inactivation is achieved, to the limit of detection of thisassay. TABLE 2 Sample # (QM) (μg/ml) Solvent Log Titer 1 0 Adsol 9.8 2 0DMSO 9.8 3 2.5 Adsol 3.25 4 5 Adsol 3.55 5 10 Adsol 1.0 6 20 Adsol 1.3 72.5 DMSO 5.2 8 5 DMSO 2.3 9 10 DMSO 2.4 10 20 DMSO 1.0

Example 2

[0114] The purpose of this example is to show that the presence of RBCdoes not significantly effect R17 inactivation by compounds and methodsof the present invention. Two different compounds were tested, QM andCompound 1, the synthesis of which is described in Example 17, below.For QM, the procedure was as follows: first, approximately 60% Htc RBCconcentrate was prepared by dilution in Adsol. The sample was againdiluted with Adsol into sterile tubes to give RBC concentrate with afinal Htc of 2%, 6%, 20% or 60% (0.5 ml final volume in each tube).

[0115] Next, an R17 stock (11.3 logs/ml in LB) was diluted 1:10 in Adsol(R17-Adsol). This stock was added (0.5 ml) to each tube to give a finalHtc of approximately 1%, 3%, 10% or 30% in 1 ml. A positive controlsample was prepared without RBC by combining 0.5 ml of R17-Adsol with0.5 ml Adsol. QM (3.4 mg) was dissolved in H₂O to reach a finalconcentration of 0.1 mg/ml. Then a 10 μl aliquot of the QM solution wasadded to each R17 sample and the samples were incubated approximately 2hours. A negative control was not treated with QM. The samples were thentitered in an R17 phage assay, as described in Example 1, above.

[0116] The results are shown in Table 3 and FIG. 2. It is clear from thedata that QM inactivates R17 in all of the Htc tested. TABLE 3 Sample #Htc (%) QM (μg/ml) Log Titer 1 0 0 8.8 9 0 1.0 2.0 10 1 1.0 4.0 11 3 1.02.1 12 10 1.0 2.6 13 30 1.0 2.4

[0117] Another experiment was performed to test the inactivation abilityof a novel compound, Compound 1. A 1:1000 dilution of R17 (stock titerwas 11.9 logs) was prepared in 25 ml packed red blood cells. To each of5 tubes was added 5 ml of this R17-packed red blood cell solution.Compound 1 was then dissolved in saline to a final concentration of 3mg/ml. The compound in solution was added to the 4 tubes as follows: thefirst tube, the control tube, received saline only; the second tubereceived 10 μg/ml of Compound 1 in saline; the third tube received 30μg/ml of Compound 1 in saline; the forth tube received 100 μg/mlCompound 1 in saline and the final tube received 300 μg/ml of Compound 1in saline. The tubes were mixed and then incubated at 4° C. overnight.The results showed R17 inactivation activity. Concentrations above 30μg/ml inactivated approximately 4 logs of R17 with a starting titer of10 logs of R17.

Example 3

[0118] This example sets forth the kinetics of R17 inactivation by QM.To measure the kinetics of inactivation, reactive QM must be quenched sothat intermediate time points provide a reliable measure of the R17inactivation at a particular time. Two methods were used here incombination to quench the reaction. First, NAC was added to samples toreact with excess QM. Second, samples were rapidly diluted into LBmedium to reduce the effective QM concentration in the sample. Thecontrol experiments described below demonstrate that this dual approachdoes effectively quench residual QM, allowing for a valid measure of thereaction kinetics to be taken.

[0119] Samples were prepared in the following manner. A dilution of R17(1:20) into Adsol was prepared: 0.15 ml phage (11.3 logs/ml)+2.85 mlAdsol. An aliquot of sterile-filtered 0.1 M NAC was thawed for use toquench the QM reaction with R17.

[0120] Tubes were then prepared for standard dilution of phage,containing appropriate volumes of LB. TABLE 4 Sample # Treatment 1 QMfirst, NAC quench at 0 min., dilute 2 QM first, NAC quench at 2 min.,dilute 3 QM first, NAC quench at 4 min, dilute 4 QM first, NAC quench at8 min, dilute 5 QM first, NAC quench at 16 min, dilute 6 QM first, NACquench at 32 min, dilute 7 QM first, NAC quench at 64 min, dilute 8 QMfirst, NAC quench at 128 min, dilute 9 first add NAC, then QM, dilute 10first add NAC, then QM, dilute at end 11 add NAC/no QM, dilute 12 addNAC/no QM, dilute at end 13 no NAC/no QM, dilute at end

[0121] A set of tubes were prepared, herein called quenching tubes,containing quenching factors (NAC and/or dilution with LB), to receivethe samples at appropriate time points. Cysteine (44 μl aliquots) wasadded to quenching tubes numbered 1-12.

[0122] QM (1.5 mg) was dissolved in Adsol (25.0 ml) to a finalconcentration of 0.1 mg/ml. Then the QM solution was diluted 100× intoAdsol: 50 μl QM solution+4.95 ml Adsol; 1 μg/ml final concentration.

[0123] Table 4 sets forth how each control and experimental sample wastreated. The controls were treated first, by placing aliquots (100 μl)of phage into quenching tubes 9-13, then immediately adding 100 μl of 1μg/ml QM to quenching tubes 9 and 10 and 200 μl Adsol to quenching tubes11 and 12. Adsol (250 μl) was added to quenching tube 13. Then samples 9and 11 were diluted into LB broth for phage assay.

[0124] The experimental samples were treated next. Phage (1.0 ml) wasremoved into a sterile 15 ml tube. QM (1.0 ml, 1.0 μg/ml) was added.This mixture was removed (by 200 μl aliquots) into quenching tubes 1-8at the following times: 0, 2, 4, 8, 16, 32, 64, and 128 minutes. Thesamples were mixed and immediately diluted into LB broth for phageassay. Finally, samples 10, 12, and 13 were diluted into LB broth forphage assay.

[0125] Results are shown in Table 5 and FIG. 3. While NAC alone does notkill R17 (compare samples #11 and #12 with sample #13), when addedbefore QM, NAC provided a substantial, but not complete protectionagainst QM inactivation (compare samples #7 and #10). The combination ofNAC and dilution resulted in almost complete quenching of QM activity(compare samples #1 and #13). QM inactivation of R17 was complete within2 hours. TABLE 5 Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 Log Titer 8.116.81 5.79 4.10 3.74 2.69 2.39 ≦2.4 8.66 6.32 8.73 8.35 8.80

Example 4

[0126] This experiment measures the loss of QM activity uponpre-incubation of drug in Adsol. It is believed that mustards react bythermally allowed pathways. They can be hydrolyzed in aqueous solution.This experiment was designed to measure loss of QM anti-viral activityin a particular aqueous solution, Adsol. Previous results have shownthat QM anti-viral activity did not decrease rapidly upon pre-incubationof the drug in Adsol. (Results not shown). A concern in thoseexperiments was the possibility of light-dependent inactivation, becausesamples were diluted into LB without making extraordinary efforts toshield ambient light, and because acridines are known to inactivate byphotodynamic effects. This experiment was repeated under conditionswhere ambient light levels were carefully controlled throughout theexperiment, in order to exclude the possibility that R17 inactivationwas due to light-mediated effects. Also, additional controls were addedto examine the effects of light in samples that were deliberatelyexposed to room lights and to examine the inactivation by the parentcompound, quinicrine, the structure of which follows:

[0127] The following procedure was performed in a biosafety cabinetwithout lights. R17 phage (10.9 logs/ml) was diluted 10 fold into Adsol:1.0 ml R17 stock+9.0 ml Adsol. One (1) ml of the diluted phage wastransferred into ten sterile 1.5 ml tubes. A 0.1 mg/ml QM solution wasprepared by dissolving 3.4 mg QM (weighed in hood) with 34 ml H₂O. Theresulting solution was wrapped in foil to shield from light. Then, 10 μlQM was added to tubes after 0, 10, 40, 60, 120 or 240 minutes ofpre-incubation. Samples were again wrapped in foil to prevent exposureto light. For a light control, 10 μl QM was added to 1.0 ml phage attime 0, and the sample was not wrapped in foil. A 1 mg/ml solution ofquinicrine in DMSO was prepared as another control. One (1) μl of thiswas added to each of two samples. Then one sample was incubated in foil(sample Q) and one without foil (sample Q+light). All samples wereincubated for 2 hours 15 minutes beyond final addition of QM. Totalincubation for the time zero sample was 6 hours 15 minutes.

[0128] The following work was performed with very low ambient light(source was one closed doorway): bacteria was diluted and plated in thedark. For the light positive controls, the samples were exposed toambient light during dilutions, then moved to dim lighting conditionsduring plating.

[0129] The results are shown in FIG. 4 and Table 6. From these resultsit is clear that there was no light-dependent kill by QM or quinicrineunder the conditions of this experiment. Further, QM activity was notdiminished after a 4 hour pre-incubation in Adsol. TABLE 6 QM + Q +Sample Control light Q light 0 min 10 min 40 min 1 hour 2 hour 4 hourLog 9.7 3.9 9.7 9.7 3.8 3.9 3.9 4.0 4.8 4.0 Titer

Example 5

[0130] QM activity was not diminished after a 4 hour pre-incubation inAdsol, as shown by Example 4, above. A goal of this example is todetermine whether QM is inactivated more rapidly by pre-incubation inthe presence of red cells. This example also examines the kinetics ofremoval of QM from red blood cell solutions by an adsorbent material, toestablish the effectiveness of a scrub technique in removing compoundscontaining a mustard group.

[0131] First, phage dilutions were prepared. R17 (11.3 log/ml stock) wasdiluted 1:10 into Adsol: 0.7 ml phage+6.3 ml Adsol. Diluted phage (0.5ml) was placed into 15 sterile 1.5 ml tubes labeled 1-15. The treatmentfor each tube is shown in Table 7. TABLE 7 Sample # Treatment Time (min)1 none — 2 QM-adsol 0 3 QM-adsol 240 4 QM-RBC 0 5 QM-RBC 15 6 QM-RBC 307 QM-RBC 60 8 QM-RBC 120 9 QM-RBC 240 10 QM-XAD 0 11 QM-XAD 15 12 QM-XAD30 13 QM-XAD 60 14 QM-XAD 120 15 QM-XAD 240

[0132] Next, QM solutions were prepared. Approximately 20 ml of packedred blood cells (PRBC) were spun down in a 50 ml conical tube at 1600rpm for 9 minutes. The volume of the pellet after spinning was 17 ml.Approximately 3 mg QM was weighed out on a weighing paper in a biosafetycabinet (actual weight was 4.5 mg). The sample was then transferred to a50 ml conical tube. The sample was dissolved in Adsol to a concentrationof 0.1 mg/ml (actual volume of Adsol was 45 ml). Next, the red bloodcell pellet was diluted 1:1 with 17 ml of the QM solution. The tubecontents were mixed gently by inversion several times. This issubsequently called the QM-RBC solution.

[0133] Amberlite XAD 16™ (a commercially available adsorbent from Sigma,St. Louis, Mo.) was weighed out (0.452 g) and transferred to a 15 mlconical tube. An aliquot of the QM-RBC solution (9 ml) was transferredto the 15 ml tube containing 0.5 g XAD-16 and mixed gently by inversion.This is subsequently referred to as the QM-XAD solution. The QM solutionwas diluted with an equal volume of Adsol (1 ml of each). This issubsequently referred to as the QM-Adsol solution.

[0134] At each time point, 0.1 ml was removed from QM-RBC, QM-XAD andQM-Adsol into a 1.5 ml eppendorf tube. The tubes' contents were spundown 10 sec at full speed in a microfuge to pellet cells and resin. Then5 μl aqueous phase containing QM was transferred to the appropriate tubecontaining phage. The phage containing QM was then incubated in the darkfor the times specified in Table 7, above. The 240 minute sample wasincubated at least 2 hours after addition of QM. Finally, dilutions weremade and the phage were plated.

[0135] The results appear in Table 8, below, and FIG. 5. QM anti-viralactivity was removed upon a 4 hour pre-incubation with red blood cells.The adsorbent scrub material, Amberlite XAD-16™, also removed QM fromblood within 1 hour. These results suggest that either incubation in thepresence of red blood cells or treatment with an adsorbent resin, or thetwo treatments combined, will be sufficient to rapidly remove residualQM after inactivation. TABLE 8 Sample Log Titer 1 9.7 2 2.9 3 3.7 4 2.85 3.4 6 5.0 7 7.1 8 8.0 9 9.7 10 3.2 11 7.9 12 9.1 13 9.6 14 9.8 15 9.8

Example 6

[0136] The purpose of this example is to measure inactivation of duckhepatitis B virus (DHBV) by a method of the present invention. DHBV waschosen as a model for human hepatitis B virus because of thesimilarities in design between the two viruses. See Ganem, D. andVarmus, H. “The Molecular Biology of the Hepatitis B Viruses,” Ann.Biochem. 56:651 (1987).

[0137] Infected duck hepatocytes were prepared as follows. Duckhepatocytes were isolated from the livers of approximately 1 week oldducklings. Ducklings were prescreened and found negative for DHBV. Eachof the ducklings was anesthetized, then infused with 0.5 ml sodiumheparin via the portal vein. Next, each duckling was perfused with 75 mlof a solution containing 200 ml 1×MEM/Earle's BSS+2 ml Hepes buffer+2 mlof 1% EGTA (in 1×MEM). Then, the ducklings were perfused for 20 minuteswith a filter sterilized solution containing 30 mg of Collagenase A(commercially available from Boehringer-Mannheim Biochem., Indianapolis,Ind.) +200 ml Ham's F-12/DMEM medium.

[0138] At this point, the liver was removed, cut up into a fine mush andplaced in a 125 ml bottle containing 50 ml Ham's F-12/DMEM.Approximately 10 ml of a solution containing 5 mg DNase I and 25 mlHam's F-12/DMEM was added to the liver suspension. The suspension wasspun at 200 rpm for 10 minutes.

[0139] The suspension was then strained through gauze pads, the 125 mlbottle was rinsed with the remaining 15 ml of the DNase I solution andthe rinsing was also strained into the liver suspension. The cellsuspension was equally divided into 2×50 ml centrifuge tubes andpelleted at 50×g for 2 minutes. The pellets were resuspended in 10 ml ofa solution containing Medium 199/Earle's BSS, 5% calf serum andpelleted. This process was repeated two more times. The third pelletingwas resuspended in 10 ml plating medium. Another 10 ml plating mediumwas added to each tube.

[0140] The liver cell suspension was filtered through a 70 micron cellstrainer into a 50 ml centrifuge tube. Aliquots of the cell suspension(approximately 0.5 ml) were transferred to petri dishes containing 2 mlplating medium, to obtain a level of confluency corresponding toapproximately 6 to 8×10⁶ viable cells per petri dish. After a two hourincubation at 37° C., the medium was changed to L-15 medium(commercially available from Gibco, Grand Island, N.Y.) (containing 0.9g/L galactose, 0.55 g/L Na pyruvate)/DMSO. The medium was again changedat 24 hours and every 48 hours thereafter. Cells were grown in culturefor 5-7 days.

[0141] Next, viral inactivation was performed. DHBV stock virus wasthawed at 37° C. for 15 min in an oven. The virus was then spun down at14000 rpm in a microfuge for 5 min at room temperature and thesupernatant was transferred to a fresh tube, avoiding material at thebottom of the tube. The spin and transfer were repeated and the sampleswere placed on ice.

[0142] Approximately 7 ml of whole blood was drawn into a tubecontaining acid citrate dextrose anticoagulant. The cells were spun downat 1600 rpm for 9 minutes to pack the red cells. The plasma waswithdrawn and replaced with an equal volume of Adsol (2.9 ml).

[0143] The virus was diluted 0.25 ml into 2.25 ml red blood cells andthe mixture was vortexed to create a 10⁻¹ dilution. The diluted viruswas then aliquoted in sterile tubes as follows: 50 μl as an untreatedsample; 1.8 μl to be treated with 40 μg/ml QM; and 0.5 ml to be treatedwith 10 μg/ml QM. Next, a 1 mg/ml QM solution was prepared by dissolving3.2 mg QM in 3.2 ml sterile ddH₂O. Aliquots of the QM solution wereadded to the tubes containing virus, as follows: 72 μl QM was added tothe 1.8 ml sample to achieve a final concentration of QM of 40 μg/ml and5 μl QM was added to the 0.5 ml sample to achieve a final concentrationof QM of 10 μg/ml. The samples were incubated for 4 hours at roomtemperature.

[0144] After incubation the red blood cells were spun down in amicrofuge. Plasma/Adsol supernatant was removed. Dilutions of eachsample were prepared by serial dilution of 100 μl virus into 0.9 mlPBS/10% NCS (PBS was 10 mM, pH 7.4). The untreated control (sample #1)was diluted to 10⁻⁷. The treated samples (#2 and #3) was diluted to10⁻⁴.

[0145] Plates containing liver suspension were then inoculated accordingto the scheme set forth in Table 9. Plates were inoculated withapproximately 100 μl virus in duplicate (see below) and cultured for 1,10, or 15 days. Samples were then analyzed by PCR and by slot blothybridization to confirm the presence of viral DNA.

[0146] Slot blot hybridization was performed for all of the samplesafter harvesting DNA from tissue culture samples. PCR analysis wasperformed on selected samples. Samples were denatured with 3M NaOH, aswere plasmid pD1.5G DNA samples for labeling. Samples were thenneutralized with NH₄OAc. 400 μl of 1M NH₄OAc was added to each well of aMini Fold II Slot Blot Apparatus, commercially available from VWRScientific, Greenbelt, Mo., fitted with a filter, as were aliquots ofeach sample. Vacuum was applied to the apparatus until all samples hadbeen pulled through the filter. The filter was then baked to dry. Next,the filter was pre-hybridized in a mixture of 250 ml of 20×SSC (175.3 gNaCl, 88.2 g Na citrate in 800 ml H₂O), 50 ml of 50×Denhardt's solution(5 g Ficoll, available from Sigma, St. Louis, Mo., 5 gpolyvinylpyrrolidone, and 5 g bovine serum albumin with 500 ml H₂O), 20ml of h mg/ml denatured salmon sperm DNA, 180 ml H₂O, 500 ml formamideand 10 ml of 10% solution of sodium dodecyl sulfate in H₂O. Probe wasprepared as follows: 3 μl of pD1.5G (67 ng/μl) and 5 ml of 15 ng/μlrandom hexamer oligonucleotides were heated and cooled again, then 4 μlof 5×labeling buffer, 2 μl of dGAT mixture (5 mM each of dGTP, dATP,dTTP, in TE), 1 μl of Klenow, and 5 μl of [a³²P]dCTP was added andincubated. Reaction was stopped by adding 25 mM EDTA. Then 5×10⁵ countsper minute of probe per ml of hybridization solution was added to thefilter and allowed to hybridize overnight. The filter was removed, andlow stringency wash solution (50 ml of 20×SSC, 940 ml of H₂O and 10 mlof 10% SDS) was added to cover the filter for a wash during shaking,which was repeated 2 times, the last time adding high stringency wassolution (5 ml of 20×SSC, 990 ml of H₂O and 10 ml of 10% SDS) instead.The filter was then exposed to film to obtain an appropriate exposure,and the film was then scored for positive hybridization. A negativecontrol sample containing calf thymus DNA was also run. TABLE 9 SampleTreatment Dilution Incubation 1 no virus NA 10 days 2 untreated 10⁻⁷ 10,15 3 untreated 10⁻⁶ 10, 15 4 untreated 10⁻⁵ 10, 15 5 untreated 10⁻⁴ 10,15 6 untreated 10⁻³ 10, 15 7 40 μg/ml QM 10⁻⁵ 10, 15 8 40 μg/ml QM 10⁻⁴10, 15 9 40 μg/ml QM 10⁻³ 10, 15 10 40 μg/ml QM 10⁻² 10, 15 11 40 μg/mlQM 10⁻¹ 1, 10, 15 12 10 μg/ml QM 10⁻⁵ 10, 15 13 10 μg/ml QM 10⁻⁴ 10, 1514 10 μg/ml QM 10⁻³ 10, 15 15 10 μg/ml QM 10⁻² 1, 10, 15

[0147] Table 10 summarizes PCR and slot blot hybridization data. (NPsignifies that PCR was “not performed” for that sample. A plus signsignifies that DHBV nucleic acid was amplified in PCR. A minus signsignifies that it was not amplified). TABLE 10 Sample # Incubation(days) Plate #'s Blot Results PCR Results 1 10 1*, 2 −, − − 2 10 3, 4 −,− NP 2 15 5*, 6 −, − − 3 10 7, 8 −, − NP 3 15 9*, 10 −, − + 4 10 11*, 12−, − − 4 15 13*, 14 ±, + + 5 10 15*, 16* +, − +, + 5 15 17*, 18 +, + + 610 19, 20* +, + + 6 15 21*, 22 +, + + 7 10 23, 24 −, − NP 7 15 25, 26 −,− NP 8 10 27, 28 −, − NP 8 15 29, 30 −, − NP 9 10 31*, 32 −, − − 9 1533*, 34 −, − − 10 10 35*, 36 −, − − 10 15 37*, 38 −, − − 11 1 39, 40* −,− − 11 10 41*, 42 −, − − 11 15 43*, 44 −, − − 12 10 45, 46 −, − NP 12 1547, 48 −, − NP 13 10 49, 50* −, − − 13 15 51, 52 −, − NP 14 10 53*, 54*−, − −, − 14 15 55, 56* −, − − 15 1 57*, 58 −, − − 15 10 59, 60* −, − +15 15 61*, 62 −, − +

[0148] Referring to Table 10, viral titer was 6 logs per ml based on PCRpositive signal for plate #9. A dose of 10 μg/ml QM inactivated 4 logsper ml based on PCR positive signal for plates #60 and #61. A dose of 40μg/ml QM inactivated>6 logs of DHBV per ml based on the absence of a PCRsignal and slot blot signals in all samples tested.

Example 7

[0149] The purpose of this example is to measure inactivation ofcell-free HIV by QM. As in the R17 assay, small aliquots of QM wereadded to stock HIV-1. The stock QM solution was prepared by dissolving3.4 mg of the compound in tissue culture media (DMEM/15% FBS) to reach afinal concentration of 0.6 mg/ml of QM. The QM was a colloidalsuspension rather than a solution at this concentration, which was usedin the experiment. Stock HIV (10^(4.2) plaque forming units/ml) was inDMEM/15% FBS. QM solution was added to aliquots of stock HIV-1 to obtaina final total sample volume of 0.5 ml, having the following finalconcentrations of QM: 3 μg/ml, 10 μg/ml, or 30 μg/ml. The 0.5 ml testaliquots were placed in 24 well polystyrene tissue culture plates. Twocontrols were prepared, one containing HIV-1 stock only, and onecontaining QM without HIV-1 stock. All samples were incubated for onehour at room temperature, then stored at −70° C. until assayed forinfectivity by a microtiter plaque assay. Aliquots for measurement ofresidual HIV infectivity in the samples treated with a compound of thepresent invention were withdrawn and cultured.

[0150] Residual HIV infectivity was assayed using an MT-2 infectivityassay. (Previously described in Hanson, C. V., Crowford-Miksza, L. andSheppard, H. W., J. Clin. Micro 28:2030 (1990)). The assay medium was85% DMEM (with a high glucose concentration) containing 200 μg ofstreptomycin, 200 U of penicillin, 50 μg of gentamicin, and 1 μg ofamphotericin B per ml, 15% FBS and 2 μg of Polybrene (Sigma ChemicalCo., St. Louis, Mo.) per ml. Test and control samples from theinactivation procedure were diluted in 50% assay medium and 50% normalhuman pooled plasma. The samples were serially diluted in 96-well plates(Corning Glass Works, Corning, N.Y.). The plates were incubated at 37°C. in a 5% CO₂ atmosphere for 1 to 18 hours. MT-2 cells (0.025 ml)[clone alpha-4, available (catalog number 237) from the NationalInstitutes of Health AIDS Research and Reference Reagent Program,Rockville, Md.] were added to each well to give a concentration of80,000 cells per well. After an additional 1 hour of incubation at 37°C. in 5% CO₂, 0.075 ml of assay medium containing 1.6% SeaPlaque agarose(FMC Bioproducts, Rockland, Me.), prewarmed to 38.5° C. was added toeach well. The plates were kept at 37° C. for a few minutes untilseveral plates had accumulated and then centrifuged in plate carriers at600×g for 20 minutes. In the centrifuge, cell monolayers formed prior togelling of the agarose layer. The plates were incubated for 6 days at37° C. in 5% CO₂ and stained by the addition of 0.05 ml of 50 μg/mlpropidium iodide (Sigma Chemical Co.) in phosphate-buffered saline (pH7.4) to each well. After 24 to 48 hours, the pink/orangefluorescence-stained microplaques were visualized by placing the plateson an 8,000 μW/cm² 304 nm UV light box (Fotodyne, Inc., New Berlin,Wis.). The plaques were counted at a magnification of 20× to 25× througha stereomicroscope. TABLE 11 Sample Log Titer no QM 4.2 3 μg/ml QM 3.410 μg/ml QM 2.0 30 μg/ml QM <1.7

[0151] The results appear in Table 11, above. At a concentration of 30μg/ml, QM was able to inactivate cell-free HIV completely to the levelof detection of the plaque assay used.

Example 8

[0152] The last example demonstrated that QM was able to inactivate cellfree HIV. HIV can also be found within certain types of cells. Thisexample examines the ability of QM, at varying concentrations, toinactivate the cell-associated form of HIV.

[0153] H9 cells chronically infected with HIV_(IIIB) were used.(H9/HTLV-III-B NIH 1983 Cat.#400). Cultures of these cells weremaintained in high glucose Dulbecco Modified Eagle Medium supplementedwith 2 mM L-glutamine, 200 units/ml penicillin, 200 μg/ml streptomycin,and 9% fetal bovine serum (Intergen Company, Purchase, N.Y.) Formaintenance, the culture was split once a week, to a density of 3×10⁵ to4×10⁵ cells/ml. About four days after splitting, 8.8% sodium bicarbonatewas added as needed. For the inactivation procedure, the cells were usedthree days after they were split. They were spun from their culturemedium at 400 g for 10 minutes, the supernatant was discarded, and thecells were resuspended in approximately 8 ml of 85% DMEM+15% FBS, to aconcentration of 2×10⁶ cells/ml. Aliquots (1 ml) of the infected cellsuspension were placed in 15 ml tubes for QM free controls and for theQM experimental sample. A stock solution of QM (1 mg/ml in sterileddH₂O) was diluted into the 15 ml tubes in the appropriate aliquots toyield a final concentration of either 0, 3, 10, 30, 100, or 150 μg/ml.The samples were incubated for two hours, with periodic thouroughmixing, then stored at −80° C. until analyzed by microtiter plaqueassay.

[0154] The stored samples were thawed at 37° C., then titrated in an HIVmicrotiter plaque assay, as described in Hanson, C. V., Crawford-Miksza,L. and Sheppard, H. W., J. Clin. Micro 28:2030 (1990), and as describedin Example 7, above, with the following modifications. The samples wereserially diluted directly in 96-well plates (Corning Glass Works,Corning, N.Y.). The plates were incubated at 37° C. in a 5% CO₂atmosphere for 1 to 18 hours. MT-2 cells (0.025 mL) [clone alpha-4,available (catalog number 237) from the National Institutes of HealthAIDS Research and Reference Reagent Program, Rockville, Md.] were addedto each well to give a concentration of 80,000 cells per well. After anadditional 1 hour of incubation at 37° C. in 5% CO₂, 0.075 mL of assaymedium containing 1.6% SeaPlaque agarose (FMC Bioproducts, Rockland,Me.), prewarmed to 38.5° C. was added to each well. The plates were keptat 37° C. for a few minutes until several plates had accumulated andthen centrifuged in plate carriers at 600×g for 20 minutes. In thecentrifuge, cell monolayers formed prior to gelling of the agaroselayer. The plates were incubated for 6 days at 37° C. in 5% CO₂ andstained by the addition of 0.05 mL of 50 μg/mL propidium iodide (SigmaChemical Co.) in phosphate-buffered saline (pH 7.4) to each well. After24 to 48 hours, the pink/orange fluorescence-stained microplaques werevisualized by placing the plates on an 8,000 μW/cm² 304 nm UV light box(Fotodyne, Inc., New Berlin, Wis.). The plaques were counted at amagnification of between 20× and 25× through a stereomicroscope.

[0155] The results appear in Table 12. TABLE 12 Sample Log Titer LogReduction no QM  5.5 — 3 μg/ml  2.7  −2.8 10 μg/ml <0.7 >−4.8 30 μg/ml<0.3 >−4.8 100 μg/ml   1.75  −3.75 150 μg/ml <0.3 >−5.2

[0156] It is clear from this example that QM inactivates cell-associatedHIV, even at very low concentrations such as 10 μg/ml and below.

Example 9

[0157] This example sets forth the ability of two compound having anucleic acid binding ligand and a mustard group, QM, andN-(2-chloroethyl)-N-ethyl-N′-(6-chloro-2-methoxy-9-acridinyl)-1,3-propanediaminedihydrochloride (“ICR-170”) (commercially available from PolysciencesInc, Warrington, Pa.) to inactivate both cell-free and cell-associatedHIV in the presence of red blood cells. The structure of ICR-170 isshown below.

[0158] For the cell free HIV inactivation, 15 ml of PRBC was mixed with5 ml Adsol for a final volume of 20 ml. Then ten 2 ml aliquots wereadded to 15 ml conical tubes. Varying doses of the two compounds werenext added to the tubes. The stock compound solutions were both 1 mg/mlin saline, stored at 4° C. ICR-170 was in solution at thisconcentration. The following volumes of the two test compounds wereadded to the PRBC tubes: 20, 40, 80 or 160 μl; to produce finalconcentrations of the test compound of 10, 20, 40, or 80 μg/ml.

[0159] After addition of the compounds, the samples were incubated for100 minutes at room temperature in the dark, with mixing every 30minutes. Subsequently, the red blood cells were pelleted by spinning for5 minutes at 2500 rpm. The supernatant was removed and NHSP was added sothat the sample contained 15% NHSP. Samples were stored at −80° C.

[0160] Inactivation of cell-associated HIV was performed in a similarmanner, with the following exceptions. H9 cells chronically infectedwith HIV_(IIIB) were used. (H9/HTLV-III-B NIH 1983 Cat.#400). Culturesof these cells were maintained in high glucose DMEM supplemented with 2mM L-glutamine, 200 units/mL penicillin, 200 μg/ml streptomycin, and 9%fetal bovine serum (Intergen Company, Purchase, N.Y.) For maintenance,the culture was split once a week, to a density of 3×10⁵ to 4×10⁵cells/ml and about four days after splitting, 3.3% sodium bicarbonatewas added as needed. For the inactivation procedure, the cells were usedthree days after they were split. The cells in a sample of this stockwere counted on a Neubauer type Hemacytometer (commercially availablefrom VWR Scientific, Greenbelt, Mo.), and found to have 1.07×10⁶cells/ml. An aliquot (18.7 ml, 20×10⁶ cells) was pelleted andresuspended in 5 ml Adsol. This 5 ml of cell suspension was then addedto 15 ml of PRBC. The sample was divided and compound was added asdescribed above for the cell-free samples. The samples were incubatedfor 100 minutes, followed by the addition of 3 ml of a 1:1 mixture ofNHSP and RPMI-1640 (commercially available from Irvine Scientific, SantaAna, Calif.). Next, each sample was placed in a 15 ml tube containing 6ml lymphocyte separation medium (LSM) (commercially available fromOrganon Teknika Corp., Durham, N.C.) and the tubes were spun at 1500 rpmfor 30 minutes. The H9 cells, which separated into a distinct layer,were removed to another tube, mixed with 10 ml DMEM and spun at 2000 rpmfor 5 minutes. The pellet was resuspended into 1 ml of 85% DMEM+15% FBS,and then transferred to a 2 ml sarstedt tube. The samples were alsostored at −80° C.

[0161] The samples were titered using a microtiter plaque assay, asdescribed in Example 8 for cell-free HIV and Example 9 forcell-associated HIV. The results appear in Table 13A (cell free) and 13B(cell associated), below. TABLE 13A Cell-free HIV Inactivation Log TiterCompound Concentration (μg/ml) pfu/ml Log Reduction QM  0 5.7 — 10 3.91.8 20 3.0 2.7 40 0 >4.3  80 0 >4.3  ICR-170  0 5.7 — 10 5.1 0.6 20 4.41.3 40 3.4 2.3 80 1.7 4.0

[0162] TABLE 13B Cell-associated HIV Inactivation Compound Concentration(μg/ml) Log Titer Log Reduction QM  0 5.5 — 10 4.4 1.1 20 3.6 1.9 40 2.53.0 80 2.4 3.1 ICR-170  0 5.7 — 10 5.2 0.5 20 4.5 1.2 40 3.7 2.0 80 3.72.0

Example 10

[0163] The above examples have established that QM has exceptionalpathogen inactivation activity. In choosing an agent to decontaminateblood products for clinical testing or transfusion, it is also importantto consider the effects of the method and compound used on blood productfunction. This example explores the short term effects of two compoundshaving a nucleic acid binding ligand and a mustard group, QM andchlorambucil on red blood cell function, as measured by potassiumleakage and IgG binding to red blood cell surfaces. The structure ofchlorambucil appears below.

[0164] This example additionally compares the R17 inactivation activity,in red blood cells, of a compound having both a nucleic acid bindingligand and a mustard group (QM), with a compound having only a mustardgroup, and no nucleic acid binding ligand (chlorambucil).

[0165] Whole blood (20 ml) was transferred to a 50 ml conical tube andspun down at 1600 rpm for 9 minutes at room temperature. Plasma wasremoved (9 ml). Next, 10.9 logs/ml stock of R17 phage was diluted 1:20with Adsol (24.4 ml Adsol+1.28 ml R17). The pelleted red blood cellswere then resuspended to 30% Htc with 25.6 ml of the Adsol/R17 mixture.Aliquots (3 ml each) were transferred into 9 tubes on ice.

[0166] Each mustard was added to Adsol. Chlorambucil, commerciallyavailable from Aldrich Inc., Milwaukee, Wis., (5.8 mg) was added to 1.93ml Adsol plus 5.85 μl 3M NaOH (undesolved material remained, andsuspension was used in the experiment by swirling before addition. QM(2.9 mg) was added to 0.967 ml Adsol, (again, material remained insuspension). The mustards were immediately added to the blood, atvolumes set forth in Table 14, below, and mixed by inversion TABLE 14Sample Contents Volume Mustard 1 control none 2 10 μg/ml Chlorambucil 10 μl 3 30 μg/ml Chlorambucil  30 μl 4 100 μg/ml Chlorambucil 103 μl 5300 μg/ml Chlorambucil 333 μl 6 10 μg/ml Quinacrine  10 μl 7 30 μg/mlQuinacrine  30 μl 8 100 μg/ml Quinacrine 103 μl 9 300 μg/ml Quinacrine333 μl

[0167] Extracellular potassium levels were measured approximately onehour after treatment using a Ciba Corning 614 K⁺/Na⁺ Analyzer(commercially available from Ciba Corning Diagnostics Corp., Medfield,Mass.). The remaining samples were incubated overnight at 4° C. Afterincubation, 0.2 ml of each sample was removed for R17 assay and spun ina microfuge for 1 min. Supernatant was then removed for phage assay.

[0168] Potassium levels on remaining samples were measured and thesamples were stored at 4° C. Potassium measurements were repeated dailyfor one week or until significant differences were observed.Extracellular potassium data appears in Table 15, and FIG. 6. IgGbinding in the samples was measured using Baxter Unival Anti-HumanGlobulin Anti-IgG for Direct Antiglobulin Test and Baxter Coombs controlCells for Quality Control of Anti-Human Globulin Test (both availablefrom Baxter Healthcare Corporation, Deerfield, Ill.). The results of IgGBinding as measured by FACScan™ (Becton Dickinson, Mountain View,Calif.) appear Tables 15 and 16:

[0169] R17 was completely inactivated at all concentrations of QM (≧8.4logs/ml). However, little or no inactivation (≦0.4 logs) was observedfor Chlorambucil, up to a concentration of 300 μg/ml. TABLE 15Extracellular Potassium mM Day Sample 0 Day 1 Day 2 Day 3 Day 4 Day 6Day 7 1 0.70 1.59 2.51 3.16 3.71 4.63 5.01 2 0.76 1.59 2.45 3.18 3.724.57 4.94 3 0.69 1.56 2.40 3.16 3.72 4.69 5.03 4 0.72 1.74 2.43 3.183.76 4.73 5.12 5 0.72 1.71 2.58 3.31 3.92 4.89 5.36 6 0.73 1.65 2.763.64 4.26 5.30 5.69 7 0.76 1.94 3.08 4.00 4.63 5.76 6.15 8 0.78 2.484.05 5.23 6.06 7.50 8.02 9 0.82 3.61 5.59 7.37 8.32 >10 11.20

[0170] TABLE 16 Sample 1 2 3 4 5 6 7 8 9 Median Fluorescence 3.43 3.313.37 3.31 3.62 5.00 7.77 15.8 48.7

[0171] Chlorambucil did not alter potassium leakage or IgG binding ofred cells. QM showed significant anti-viral activity and only inducedslight changes in red blood cell function under the conditions of thisexperiment. Significant red blood cell damage was only detected atlevels far higher than that required to inactivate R17.

Example 11

[0172] Example 10 showed that QM was able to inactivate R17 in red bloodcells under conditions where potassium leakage and surface IgG bindingwere negligible. This example is designed to further these observationsby looking more extensively at red blood cell function after treatmentwith varying levels of QM. Specifically, this example looks at theeffects of QM treatment on red blood cell function after storage underconditions that closely mimic those in a blood bank.

[0173] A packed red blood cell unit, approximately 1 day old, wasobtained from Sacramento Blood Center. The cells were resuspended andapproximately 200 ml was transferred to a sterile container. R17 (0.2ml) in LB was added and the sample was mixed. Next the unit was dividedinto 6-30 ml aliquots in sterile conical centrifuge tubes on ice. Theremaining packed red blood cells were stored in the bag at 4° C.

[0174] QM (3.2 mg) was mixed with ice cold Adsol (1.6 ml) to make a 2.0mg/ml suspension. Aliquots of the QM suspension were added to the cellsas set forth in Table 17. The samples were mixed thoroughly by gentleinversion and transferred to Fenwal transfer packs (Baxter/Fenwal, Ill.)for storage at 4° C. TABLE 17 Final Concentration Sample of QM (μg/ml)Volume of QM 1 0 0 2 2.5 37.5 μl 3 5   75 μl 4 10 0.15 ml 5 20 0.30 ml 640 0.60 ml

[0175] The following measurements of cell function were taken. 1)Potassium levels were determined daily for one week and weeklythereafter, using the Ciba Corning 614 K⁺/Na⁺ analyzer (commerciallyavailable from Ciba Corning, Mass.). 2) Adenosine-5′-triphosphate (ATP)and 2,3-diphosphoglyceric acid (2,3-DPG) were measured the first dayafter treatment and weekly thereafter. ATP was measured using a SigmaATP Kit, commercially available from Sigma, St. Louis Mo., followingSigma Procedure No. 366-UV hereby incorporated by reference. 2,3-DPG wasmeasured using the 2,3-DPG Kit, commercially available from Sigma, St.Louis, Mo. 3) IgG binding to the red blood cell surface was measuredafter day 1 and week 1, using the Baxter Unival Anti-Human GlobulinAnti-IgG for Direct Antiglobulin Test and Baxter Coombs Control Cellsfor Quality Control of Anti-Human Globulin Test, commercially availablefrom Baxter Healthcare, Inc., Deerfield, Ill.

[0176] The results for R17 inactivation appear in FIG. 7. The resultsfor red blood cell function appear in Tables 18A -18D. TABLE 18A QM K+K+ K+ K+ K+ K+ Concentration Day 1 Day 2 Day 7 Day 8 Day 9 Day 16control 7.41 7.41 13.20 12.96 15.32 31.72 2.5 μg/ml  7.42 7.42 12.9013.64 15.86 31.76  5 μg/ml 7.24 7.24 13.28 14.82 15.18 32.96 10 μg/ml7.24 7.24 13.06 15.16 14.68 31.64 20 μg/ml 7.00 7.00 12.90 15.38 14.4431.36 40 μg/ml 6.95 6.95 12.88 12.44 15.44 30.92

[0177] TABLE 18B QM ATP (mM) ATP (mM) ATP (mM) Concentration Day 1 Day 9Day 16 control 0.77 0.80 0.75 2.5 μg/ml 0.78 0.80 0.74   5 μg/ml 0.780.81 0.73  10 μg/ml 0.76 0.81 0.73  20 μg/ml 0.78 0.80 0.73  40 μg/ml0.76 0.80 0.72

[0178] TABLE 18C QM 2,3-DPG 2,3-DPG 2,3-DPG Concentration Day 1 Day 9Day 16 control 2.20 0.77 0.89 2.5 μg/ml 2.20 0.88 0.10   5 μg/ml 2.350.97 0.28  10 μg/ml 2.06 1.11 0.34  20 μg/ml 2.63 1.43 1.36  40 μg/ml2.09 1.04 0.11

[0179] TABLE 18D QM mean FL median FL mean FL median FL mean FL medianFL Concentration Day 1 Day 1 Day 9 Day 9 Day 16 Day 16 control 4.41 4.14.41 4.1 4.41 4.1 2.5 μg/ml  4.77 4.45 4.77 4.45 4.77 4.45  5 μg/ml 4.794.45 4.79 4.45 4.79 4.45 10 μg/ml 4.96 4.7 4.96 4.7 4.96 4.7 20 μg/ml5.73 5.19 5.73 5.19 5.73 5.19 40 μg/ml 6.31 6.04 6.31 6.04 6.31 6.04

[0180] Under conditions of effective R17 inactivation in packed redblood cells, there are no significant effects on potassium-leakage, ATPcontent or 2,3-DPG content, and only modest effects on IgG binding toRBCs.

Example 12

[0181] This example evaluates QM to determine whether it is mutagenic inthe Ames test, a well known assay for mutagenicity. While mustards areproving to be effective compounds for pathogen inactivation, they arealso considered potential mutagens. This example shows that bloodtreated with QM does not exhibit significant mutagenic action,particularly after an incubation period. Thus, the compounds of thepresent invention have exceptional pathogen inactivation efficiencywhile displaying only minimal mutagenicity.

[0182] In this example QM was tested for its mutagenicity using an Amesassay. The mutagenicity was tested under four conditions: QM incubatedovernight in water, QM added to red blood cells and immediately plated;QM added to red blood cells, incubated overnight at 4° C. and thenplated; and QM added to red blood cells, incubated 4 hours at 4° C.,then mixed with Amberlite XAD-16™ and incubated overnight beforeplating.

[0183] First, the solubility of QM in red blood cells was determined. 10mg/ml QM was diluted 10-fold and 100-fold into red blood cells and thesolubility was observed. To obtain a 1.0 mg/ml solution, 20 μl of the 10mg/ml QM solution was combined with 180 μl of the 50% Htc red bloodcells. This stock contained definite particles. Next a 0.3 mg/mlconcentration was tested by combining 20 μl of 3.0 mg/ml QM and 180 μlof 50% Htc red blood cells. There was evidence of precipitating out ofsolution with the 3 mg/ml stock. Finally, 20 μl of the 1.0 mg/ml stockwas mixed with 180 μl of packed red blood cells. The 1.0 mg/ml stockappeared clear. The 3 mg/ml stock was chosen as the highestconcentration, thus 0.3 mg/ml in red blood cells and 30 μg/plate are theupper concentration limits in this experiment.

[0184] Preparation of these three test mixtures was as follows. A 10mg/ml solution of QM in DMSO was diluted to 1.0 mg/ml (60 μl of QMsolution added to 0.54 ml DMSO). A 50% Htc red blood cell solution wasprepared by spinning down 10 ml of a packed red cell unit at 1600 rpmfor 9 minutes. Supernatant was removed and the cell pellet wasresuspended in an equal volume of Adsol. Htc was then confirmed on aModel F800 Sysmex cell counter (Toa Medical Electronics, Kobe, Japan).Thirteen 0.9 ml aliquots of RBC solution were then placed in test tubes.Four different stock solutions of QM were prepared because the compoundmay precipitate out of solution at concentrations as low as 3 mg/ml.Stock solutions at varying concentrations were prepared by making thefollowing dilutions of a 1.0 mg/ml solution: 150 μl of a 1.0 mg/ml QMsolution+350 μl DMSO to produce a 0.3 mg/ml solution; 40 μl of a 1.0mg/ml QM solution+360 μl DMSO to produce a 0.1 mg/ml solution; 15 μl ofa 1.0 mg/ml QM solution+485 μl DMSO to produce a 0.03 mg/ml solution. Tothe first tube, 100 μl DMSO was added and the tube was placed on a 4° C.shaker (25 rpm, Orbital Shaker, commercially available from VWRScientific, Greenbelt, Mo.) for overnight incubation. Tubes 2-5 wereshaken overnight as well, then 100 μl aliquots of each QM solution wasdiluted into the tubes just before addition to the Ames strains. Totubes 6-9 was added 100 μl of each QM solution. The tubes were thenincubated overnight at 4° C. on the shaker. Finally, 100 μl of each QMsolution was also added to tubes 10-13, which were then incubated on theshaker for 4 hours. Subsequently, 0.1 g of a polymeric adsorbentmaterial, Amberlite XAD 16™ (commercially available from Sigma, SaintLouis, Mo.), was added to each of tubes 10-13 and the incubation wascontinued overnight. The final contents of each tube, and the stock QMsolutions used, are listed in Table 19, below. TABLE 19 QM STOCK SAMPLENUMBER CONTENTS SOLUTION  1 RBC + DMSO none  2 RBC + 0.003 mg/ml QM 0.03mg/ml  3 RBC + 0.01 mg/ml QM 0.1 mg/ml  4 RBC + 0.03 mg/ml QM 0.3 mg/ml 5 RBC + 0.1 mg/ml QM 1 mg/ml  6 RBC + 0.003 mg/ml QM 0.03 mg/ml  7RBC + 0.01 mg/ml QM 0.1 mg/ml  8 RBC + 0.03 mg/ml QM 0.3 mg/ml  9 RBC +0.1 mg/ml QM 1 mg/ml 10 RBC + 0.003 mg/ml QM 0.03 mg/ml 11 RBC + 0.01mg/ml QM 0.1 mg/ml 12 RBC + 0.03 mg/ml QM 0.3 mg/ml 13 RBC + 0.1 mg/mlQM 1 mg/ml

[0185] In a separate experiment, samples of QM in water were prepared asfollows. Sample tubes were labeled and 0.5 ml phosphate buffer was addedto each one. Then various dilutions of a stock solution of QM (1 mg/ml)were added to five of the tubes. For 100 μg/plate−1.4 ml stock solution;for 30 μg/plate−0.42 ml stock+0.98 ml H₂O; for 10 μg/plate−0.14 milstock+1.26 ml H₂O; for 3 μg/plate−0.042 ml stock+1.358 ml H₂O; for 1μg/plate−0.014 ml stock+1.386 ml H₂O. A control was also prepared usingonly H₂O.

[0186] The procedures used for the Salmonella mutagenicity test asdescribed in detail by Maron and Ames were followed exactly. Maron, D.M. and B. N. Ames, Mutation Research 113: 173 ( 1983). A briefdescription for each procedure is given here. The tester strains TA97a,TA98, TA100, TA102, TA1537 and TA1538 were obtained from Dr. Ames.TA97a, TA98, TA1537 and TA1538 are frame shift tester strains. TA100 andTA102 are base-substitution tester strains. Upon receipt each strain wascultured under a variety of conditions to confirm the genotypes specificto the strains.

[0187] The standard Salmonella tester strains used in this study requirehistidine for growth since each tester strain contains a different typeof mutation in the histidine operon. In addition to the histidinemutation, these tester strains contain other mutations, described below,that greatly increase their ability to detect mutagen.

[0188] Histidine Dependence: The requirement for histidine was tested bystreaking each strain first on a minimal glucose plate supplemented onlywith biotin and then on a minimal glucose plate supplemented with biotinand histidine. All strains grew only on the histidine/biotinsupplemented plates, confirming a histidine requirement.

[0189] rfa Mutation: A mutation which causes partial loss of thelipopolysaccharide barrier that coats the surface of the bacteria thusincreasing permeability to large molecules was confirmed by exposing astreaked nutrient agar plate coated with the tester strain to crystalviolet. First 100 μL of each culture was added to 2 mL of molten minimaltop agar and poured onto a nutrient agar plate. Then a sterile filterpaper disc saturated with crystal violet was placed at the center ofeach plate. After 16 hours of incubation at 37° C. the plates werescored and a clear zone of no bacterial growth was found around thedisc, confirming the rfa mutation.

[0190] uvrB Mutation: Three strains used in this study contain adeficient UV repair system (TA97a, TA98, TA100, TA1537 and TA1538). Thistrait was tested for by streaking the strains on a nutrient agar plate,covering half of the plate, and irradiating the exposed side of theplate with germicidal lamps. After incubation growth was only seen onthe side of the plate shielded from UV irradiation.

[0191] R-factor: The tester strains (TA97a, TA98, TA100, and TA102)contain the pKM101 plasmid that increases their sensitivity to mutagens.The plasmid also confers resistance to ampicillin to the bacteria. Thiswas confirmed by growing the strains in the presence of ampicillin.

[0192] pAQ1: Strain TA102 also contains the pAQ1 plasmid that furtherenhances its sensitivity to mutagens. This plasmid also codes fortetracycline resistance. To test for the presence of this plasmid TA102was streaked on a minimal glucose plate containing histidine, biotin,and tetracycline. The plate was incubated for 16 hours at 37° C. Thestrain showed normal growth indicating the presence of the pAQ1 plasmid.

[0193] The same cultures used for the genotype testing were againcultured and aliquots were frozen under controlled conditions. Thecultures were again tested for genotype to confirm the fidelity of thegenotype upon manipulation in preparing the frozen permanents.

[0194] The first tests done with the strains were to determine the rangeof spontaneous reversion for each of the strains. With each mutagenicityexperiment the spontaneous reversion of the tester strains to histidineindependence was measured and expressed as the number of spontaneousrevertants per plate. This served as the background controls. A positivemutagenesis control was included for each tester strain by using adiagnostic mutagen suitable for that strain (2-aminofluorene at 5mg/plate for TA98; sodium azide at 1.5 mg/plate for TA100;9-aminoacridine for TA 1537).

[0195] For all experiments, the pre-incubation procedure was used. Inthis procedure one vial of each tester strain was thawed and tubes wereprepared for each strain, containing 20 μL of the culture and 6 mL ofOxoid Nutrient Broth #2. This solution was allowed to shake for 10 hoursat 37° C. In the pre-incubation procedure, for each tester strain usedto evaluate the test solution, 0.1 mL of the overnight culture was addedto each of 13 sterile test tubes. To each of the tubes, 0.1 mL of thetest solution from tubes 1-13 was added. This was also performed on thesamples containing QM in water only. Then 0.5 mL of 0.2 M sodiumphosphate buffer, pH 7.4 was added. The 0.7 mL mixture was vortexed andthen pre-incubated while shaking for 20 minutes at 37° C. After shaking,2 mL of molten top agar supplemented with histidine and biotin wereadded to the 0.7 mL mixture and immediately poured onto a minimalglucose agar plate (volume of base agar was 20 mL). The top agar wasallowed 30 minutes to solidify and then the plates were inverted andincubated for 44 hours at 37° C. After incubation, the number ofrevertant colonies on each plate was counted.

[0196] The results appear in FIG. 8. Although the QM registered apositive response in the Ames test without incubation in red blood cellsand in water, an overnight incubation in red blood cells significantlyreduced the level of revertants, as did an incubation with adsorbentmaterial. A parallel experiment was performed using an activatedcharcoal adsorbent material, Hemosorba (commercially available fromAsahi Medical Corp., Tokyo, Japan.) The results, which are not shown,were similar to the results using Amberlite XAD 16™.

Example 13

[0197] As discussed above, a method of decontaminating clinical sampleswould be most useful if while it decontaminated samples, it did notsignificantly effect the results of the clinical tests themselves. Thisexample compares results of a common blood chemistry panel for samplestreated by methods of the present invention to untreated samples.

[0198] Solutions of QM and ICR-170 (2 mg/ml) were prepared in saline.The QM was almost completely dissolved, and ICR-170 remained asuspension. Next, human whole blood was drawn and 10 ml aliquots wereplaced in eight tubes. QM or ICR-170 was added to six of the tubes inaliquots of 100, 200, or 400 μl, to reach final concentrations of thecompounds of either 20, 40, or 80 μg/ml. Saline was added to theremaining two tubes, in aliquots of 100 or 400 μl, to prepare controlsamples. The samples were then allowed to clot for 30 minutes, followedby 20 minutes on a centrifuge at 1000 rpm. The separated serum was thentransferred to labeled plastic tubes and tested in a panel of 24 commonclinical chemistry tests.

[0199] The results appear below, in Table 20. Neither QM nor ICR-170 hada significant effect on the results of any of the panel of 24 clinicalchemistry tests. Lactate dehydrogenase and GGT exhibited a small drop inthe sample containing the highest concentration of ICR-170. Clearly, themethods of the present invention do not interfere significantly withclinical testing of blood samples. TABLE 20 100 40 μl 20 40 60 20 μg/ml60 salin 400 μl μg/m μg/m μg/ml μg/ml ICR17 μg/ml TEST e saline 1 QM 1QM QM ICR170 0 ICR170 Glucose 95 92 93 93 90 94 92 90 BUN 17 17 17 17 1717 17 17 Creatinine 1.1 1.1 1.1 1.2 1.1 1 1.1 1.1 Bun/Creat. ratio 15 1515 14 15 17 15 15 Sodium 143 143 138 144 146 143 144 144 Potassium 4 3.94 3.9 3.9 4 4.1 4.1 Chloride 104 106 104 106 107 103 104 103 Magnesium1.7 1.6 1.6 1.6 1.6 1.7 1.6 1.5 Calcium 9.3 8.8 9.3 8.8 9 9.4 9.2 9.1Phosphorous 4.3 3.9 4.1 4.1 4.1 4.3 4.3 4.1 inorganic protein, total 7.37.1 7.4 7.3 7.1 7.3 7.3 7.1 albumin 4.6 4.5 4.6 4.6 4.4 4.6 4.6 4.4globulin, total 2.7 2.6 2.8 2.7 2.7 2.7 2.7 2.7 A/G ratio 1.7 1.7 1.61.7 1.6 1.7 1.7 1.6 billirubin 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 alk 55 5048 50 51 54 51 47 LDH 136 134 121 125 138 129 138 151 GGT 23 21 22 22 2223 20 16 AST 17 16 18 16 16 16 17 16 ALT 20 19 17 19 18 19 19 19 UricAcid 5.6 5.3 5.5 5.4 5.4 5.7 5.6 5.5 Iron 119 107 117 113 115 119 122120 Triglycerides 164 152 159 158 158 161 150 156 Cholesterol 234 221227 226 223 231 231 223

Example 14

[0200] This example describes the inactivation of bacterial pathogens ofbiological compositions using methods of the present invention. Thefollowing experiment was performed to support that the methods of thepresent invention can be used to inactivate bacterial pathogens. In thisexample, the decontamination methods of the present invention wereapplied to inactivate Staphylococcus epidermis.

[0201] An overnight culture of the organism was made by inoculating 3 mlof LB broth from a motility stab. This was maintained at 35° C. and 1.0ml of it was used to inoculate 9 ml of LB broth in a 15 ml conical tube.A sample (1 ml) was taken for an OD₆₀₀ reading. To a tube, 5 ml of LBbroth was added. Then 50 μl of 10⁸ cfu/ml S. epidermis was added.Aliquots of the sample (1 ml each) were placed in 5 tubes. These weretreated with either 0, 3, 10, 30, or 100 μg/ml of QM. A 2 mg/ml stock ofQM in ddH₂O was added in the following amounts to produce the desiredconcentrations: 0 μl, 1.5 μl, 5 μl, 15 μl, and 50 μl. The samples wereincubated on ice for three hours.

[0202] After incubation, bacteria was quantified by plating 0.1 ml ofserial 10-fold dilutions in LB broth onto 100 mm petri dishes containingagar. After 24 hr incubation at 35° C., colonies were counted andbacterial concentration was calculated on a per ml basis. The results,which appear in FIG. 9, show that QM at >10 μg/ml inactivates S.epidermis to the level of detection of this assay.

Example 15

[0203] If a decontaminated blood product is to have value as an in vivotherapy, the blood product must retain some efficacy after thedecontamination process. One way to confirm the efficacy of a particularsample of red blood cells is to ensure that they are not cleared by therecipient's body significantly sooner than normal red blood cells whentransfused into a mammal. In this example, packed red blood cells aretreated with compounds having a nucleic acid binding ligand and amustard group, according to the methods of the present invention,transfused into mice, and tracked for post transfusion survival.

[0204] Blood was drawn from 8 Balb/c mice using an anticoagulant(containing citrate, ethalene diamine tetraacetic acid, prostaglandin E1and theophyllin) for a total of 12 ml (8 ml whole blood and 4 mlanticoagulant). An equivalent volume of Adsol was added and the samplewas centrifuged at 2000 rpm for 5 minutes. The supernatant was removedand saved as “washed solution.” The red blood cell pellet wasresuspended in Adsol to make a 50% Htc solution. Three 1.5 ml aliquotswere transferred to 14 ml round bottom polypropylene tubes.

[0205] Next, 1 mg/ml solutions of two compounds, QM and ICR-170, wereprepared in saline. The compounds were added to the three polypropylenetubes as follows: tube 1 received no treatment (120 μl of saline wasadded); tube 2 received 120 μl of the 1 mg/ml solution of QM for a finalconcentration of 80 μg/ml; tube 3 120 μl of the 1 mg/ml solution ofICR-170, for a final concentration of 80 μg/ml.

[0206] The samples were then incubated for 2 hours at 4° C. Cells werewashed three times, each time by adding 6 ml Adsol to each tube andspinning the samples at 1800 rpm for 5 minutes. After the final wash,the pellet was resuspended in Adsol buffer to a concentration of 4×10⁶cells/μl. 50 μl of each sample was removed for an unstained control.

[0207] The remaining cells were then stained with PKH26 dye. From a 1 mMPKH26 stock, 120 μl was removed and diluted with 8 ml diluent A tocreate a working solution of 15.7 μM PKH26. This solution was stored inthe dark until use. To each 2 ml of cells, 2 ml of PKH26 dye was added.The samples were mixed gently and incubated for 5 minutes at roomtemperature in the dark. The cells were remixed after 2.5 minutes. Afteranother 5 minute incubation, 2 volumes of the reserved “washed solution”was added to stop the staining reaction. The cells were centrifuged at1800 rpm for 5 minutes to pellet and the supernatant was removed. Thecells were then washed 3 times with Adsol buffer, as before. After thefinal wash, the sample volume was restored to 1 ml with Adsol. Analiquot of each sample was removed at this point for a positive stainedcontrol sample and counted on the Sysmex machine.

[0208] Swiss Webster mice were transfused with 0.2 ml of the labeledcells from each of samples 1-3 via tail vein injection. The mice werethen weighed to calculate the blood volume. Blood volume is calculatedas the animal weight in gm×0.06. Then, blood was drawn from the mice byretroorbital venipuncture using heparin-EDTA coated capillary tubes at 1hour, 24 hours, 2 more times during the first week, and one time weeklyfor 3 weeks. The eye bleeding samples were drained into isotonicsolution before analysis.

[0209] Samples were analyzed on a FACScan™ at the FL2 (red fluorescentchannel) with gating on the red cell population using forward and sidescatter linear mode gates. The proportion of labeled cells in 100,000total red cell gated events was determined. TABLE 21 SAMPLE RECOVERYLOSS PER DAY Untreated 90.3 ± 3.2 2.66% QM 80.8 ± 4.7 2.77% ICR-170 86.0± 6.6 2.71%

[0210] The results appear in Table 21. According to the results,regardless of treatment, treated cells survived in vivo as well asuntreated control cells.

[0211] Some hemolysis of RBC was detected after labeling with PKH26.Thus, recoveries may be effected by the labeling technique. Analternative labeling technique was also used, as described. An activatedbiotin ester was injected via the tail vein of the Balb/c mice in orderto label the red cells in vivo (each mouse received either a “low dosetreatment”—0.1 mg injection on two successive days, or a “high dosetreatment”—0.3 mg of biotin on three successive days). After treatment,low and high dose treatment cells with fluorescently tagged streptavidinwere clearly distinguishable as detected by FACScan analysis. Low andhigh dose treatment cells were independently treated with 80 μg/ml of QMor ICR-170 as described above. After treatment, cells were washed, mixedwith untreated cells that had been differentially labeled. Then threemice were transfused: one received untreated low and untreated high doseRBC, one received QM treated low and untreated high dose RBC, and onereceived ICR-170 treated low and untreated high RBC. Bleeding wasperformed as described above. The results appear in Table 22, below.Clearly, recovery of the treated cells is very similar to the untreatedcells. TABLE 22 % RECOVERY, LOW % RECOVERY, HIGH untreated 96.2 93.5 QM94.6 91.2 ICR-170 93.1 90.4

Example 16

[0212] Nucleic acid specific alkylating agents tested here have beenprepared by methods described in [Peck, R. M., Preston, R. K., Creech,H. J., J. Amer Chem. Soc (1959) 81, 3984; Preston, R. K., Peck, R. M.,Breuniger, E., R., Miller, A. J., Creech, H. J., J. Med. Chem. (1964) 7,471] TABLE 23 Anti-viral Activity of nucleic acid specific alkylatingagents, ACRIDINE RING - LINKING CHAIN - N(CH2CH2Cl)2

Cmpd Ring linker R17^(a) CA-HIV^(b) CF-HIV^(b) 2 A (CH2)2 + 3 A (CH2)3++ + + 4 B (CH2)3 + + 5 A (CH2)4 ++++ ++++ +++ 1 A CH(CH3)(CH2)3 +++++++ +++ 6 A (CH2)5 ++++ ++++ +++

[0213] This example sets forth the results of several screens for viralinactivation performed compounds of the present invention which haveacridine groups as their nucleic acid bining ligands. Descriptions ofhow these screens were performed are found in the above examplesdiscussing R-17, cell free HIV associated HIV. Table 23, below, showsthe results. Activity is set forth by + for lowest, up to ++++ forcomplete inactivation (to the level of detection used).

[0214] It is clear from these data that the acridine based compounds allhave inactivation action against all pathogens tested, including cellfree, cell associated HIV and R-17. 7 B (CH2)5 ++++ ++++ 8 C (CH2)5 + ++9 A (CH2)6 ++++ +++ ++ 10 B (CH2)6 ++++ +++ 11 A CH2CONH(CH2)3 ++++ +++++ 12 A CH(CH3)CONH(CH2) ++++ +++ + 2 13 A CH(CH3)CONH(CH2) ++++ +++ + 3

Example 17

[0215] This example describes the synthesis of a compound of the presentinvention, 5-[N,N-bis(2-chloroethyl)amino]methyl-8-methoxypsoralenhydrochloride (referred to throughout the text as “Compound 1”).

[0216] Step 1: The synthesis of 5-Bromomethyl-8-methoxypsoralen

[0217] To a solution of 2.69 g (12.3 mmol) of 8-methoxypsoralen(commercially available from Aldrich, Milwaukee, Wis.) in 135 mL ofglacial acetic acid was added 11 mL of bromomethyl methyl ether. Thesolution was swirled, then left for three days at room temperatureduring which a white solid precipitated. The mixture was cooled in anice bath and filtered. To the filtrate was added an additional 2.75 g(12.7 mmol) of 8-methoxypsoralen and 5 mL of BrCH₂OCH₃. After againsitting for three days, product isolation was repeated. The filter cakeswere washed with cold glacial acetic acid, air dried and finally vacuumdried to give a total yield of 6.3 g (81%) of5-bromomethyl-8-methoxypsoralen as a pale yellow solid. NMR (CDCl3):4.33 (S, 3H), 4.88 (s, 2H), 6.52 (d, J=10 Hz, 1H), 6.95 (d, J=2 Hz, 1H),7.77 (d, J=2 Hz, 1H), 8.11 (d, J=10 Hz, 1H).

[0218] Step 2: The Synthesis of5-[N,N-Bis-(2-hydroxyethyl)amino]methyl-8-methoxypsoralen

[0219] 5-Bromomethyl-8-methoxypsoralen (0.50 g, 1.6 mmol, from Step 1,above) and diethanolamine (2.56 mL, 27 mmol) were combined in 23 mL ofabsolute ethanol and refluxed for 10 hours. The solution wasconcentrated, then CHCl₃ (65 mL) was added to the residue. The organiclayer was washed with 30,30 and 10 mL of water sequentially and thecombined aqueous solutions were back extracted with CHCl₃. The combinedorganic solutions were then extracted three times, each time with 7 mLof 1.2 N HCl. The combined acid solution was taken to pH5-6 with 10%aqueous NaOH and the resultant turbid solution was washed 4 times, eachtime with 20 mL of CHCl₃. This last organic solution was rinsed with2×20 mL of brine, dried (Na₂SO₄) and concentrated to give 0.43 g (79%)of the aminediol,5-[N,N-bis-(2-hydroxyethyl)amino]methyl-8-methoxypsoralen, melting point121-122° C.; NMR (CDCl3): 2.71 (t, J=5 Hz, 4H), 3.61 (t, J=5 Hz, 4 H),4.09 (s, 2H), 4.29 (s, 3H), 6.41 (d, J=10 Hz, 1H), 7.00 (d, J=2 Hz, 1H),7.70 (d, J=2 Hz, 1H), 8.38 (d, J=10 Hz, 1H).

[0220] Step 3: 5-[N,N-Bis(2-chlorethyl)amino]methyl-8-methoxypsoralenhydrochloride

[0221] 5-[N,N-Bis(2-hydroxyethyl)amino]methyl-8-methoxypsoralen (0.030g, 0.090 mmol) was dissolved in 1 ml thionyl chloride. It was coveredwith a serum cap with a small needle vent and allowed to stir for 3days. The reaction mixture was stripped and the crude solid wasrecrystallized in isopropanol to give5-[N,N-bis(2-chloroethyl)amino]methyl-8-methoxypsoralen hydrochloride(0.012 g, 32.4%) as an off-white solid, mp 158-162° C. ¹HNMR (CD₃OD):3.40 (t, J=6 Hz, 4H), 3.86 (t, J=6 Hz, 4H), 4.33 (s, 3H), 4.70 (s, 2H),6.52 (d, J=10 Hz, 1H), 7.28 (d, J=2 Hz, 1H), 8.03 (d, J=2 Hz, 1H), 8.48(d, J=10 Hz, 1H). The chemical shift appeared to be sensitive to traceacid present.

[0222] A portion of the above salt was partitioned between methylenechloride and aqueous NaHCO₃. The organic layer was again washed withaqueous bicarbonate, dried with brine, then dried with anhydrous Na₂SO₄and evaporated to give the neutral amine; mass spectrum (EI, m/e): 371(3), 369 (4), 230 (16), 229 (100), 214 (5), 201 (5), 186 (10) (obtainedon a Shimadzu QP5000 GC/MS, with Rtx-5, 15 m column, commerciallyavailable from Shimadzu Corporation, Kyoto, Japan).

Example 18

[0223] This example describes a contemplated embodiment wherein redblood cells are treated by a method of the present invention. Thestandard blood product separation approach used presently in blood banksis as follows: three bags are integrated by flexible tubing to create ablood transfer set (e.g., commercially available from Baxter, Deerfield,Ill.). After blood is drawn into the first bag, the entire set isprocessed by centrifugation (e.g., Sorvall™ swing bucket centrifuge,Dupont), resulting in packed red cells and platelet rich plasma in thefirst bag. The plasma is expressed off of the first bag (e.g., using aFenwall™ device for plasma expression), through the tubing and into thesecond bag. The first bag, containing packed red cells, is thendetached.

[0224] In one embodiment of the decontamination approach of the presentinvention applied specifically to red blood cells, a compound having anucleic acid binding ligand and a mustard group is introduced to the redblood cells (e.g. the compound may be present in the first bag beforeblood is drawn, or transferred to the first bag after centrifugation)and incubated. After incubation, the compound may be removed using anadsorbent material (e.g., a commercially available material, such asactivated charcoal or an Amberlite resin). The adsorbent may beintroduced directly into the bag containing the red blood cells, or thered blood cells may be passed through a scrub device which contains theadsorbent. The incubation, scrub, and any subsequent storage, may takeplace in a commercially available storage bag.

[0225] From the above, it should be evident that the present inventionprovides methods of decontamination of blood preparations intended forstorage and in vivo use.

Example 19

[0226] This example describes the synthesis of 8-[3-(Bis-2-chloroethyl)amino]propyloxypsoralen (compound 5)

[0227] Step 1: 8-(3-Bromopropyloxy)psoralen

[0228] 1,3-Dibromopropane (7 mL, 68.9 mmol) was added to a slurry of8-hydroxypsoralen*ref (1.00 g, 4.95 mmol) in acetone (100 mL). After theaddition of powdered K₂CO₃ (3.0 g, 21.7 mmol, 325 mesh), the mixture wasrefluxed for 6 h, allowed to cool to room temperature, and filtered toremove K₂CO₃. The salt was rinsed with CH₂Cl₂ and the combined filtrateswere concentrated. To remove residual dibromopropane the precipitate wastriturated with hexane, then recrystallized in methylcyclohexane to give8-(3-bromopropoxy)psoralen (1.19 g, 56.9% yield), as a beige solid. ¹HNMR (CDCl₃): d 2.41 (quintet, J=5.9 Hz, 2H), 3.78 (t, J=6.5 Hz, 2H),4.64 (t, J=5.8 Hz, 2H), 6.38 (d, J=9.6 Hz, 1H), 6.82 (d, J=2.2 Hz, 1H),7.71 (d, J=2.2 Hz, 1H), 7.78 (d, J=9.6 Hz, 1H).

[0229] Step 2: 8-[3-(Bis-2-hydroxyethyl)amino]propyloxypsoralen

[0230] 8-(3-Bromopropoxy)psoralen (0.500 g, 1.55 mmol) anddiethanolamine (1.5 mL, 15.6 mmol) were refluxed in ethanol (20 mL)overnight. After rotovapping off the solvent, the resulting syrup wasdissolved in CH₂Cl₂, washed with water several times, then brine, driedwith anhydrous Na₂SO₄ and stripped of solvent to give8-[3-(bis-2-hydroxyethyl)amino]propyloxypsoralen (0.499 g, 92.9% yield),as a brown syrup. ¹H NMR (CDCl3): d 2.04 (quintet, J=6.2 Hz, 2H), 2.72(t, J=5.2 Hz, 4H), 2.91 (t, J=6.6 Hz, 2H), 3.70 (t, J=5.3 Hz, 4H), 4.54(t, J=5.7 Hz, 2H), 6.38 (d, J=9.6 Hz, 1H), 6.82 (d, J=2.1 Hz, 1H), 7.39(s, 1H), 7.70 (d, J=2.1 Hz, 1H), 7.79 (d, J=9.5 Hz, 1H).

[0231] Step 3: 8-[3-(Bis-2-chloroethyl)amino]propyloxypsoralen,(compound 5)

[0232] Thionyl chloride (0.030 mL, 0.41 mmol) was added dropwise to anice bath chilled solution of8-[3-(Bis-2-hydroxyethyl)amino]propyloxypsoralen (20.0 mg, 0.0575 mmol)and pyridine (0.016 mL, 0.21 mmol) in benzene (2 mL) and CH₂Cl₂ (2 mL).The reaction mix was covered with a serum cap and allowed to stirovernight at rt, then stripped under reduced vacuum and partitionedbetween CH₂Cl₂ and 10% NaHCO₃. The organic layer was washed severaltimes with aqueous NaHCO₃, then brine. After drying with anhydrousNa₂SO₄, solvent was rotovapped off to give8-[3-(bis-2-chloroethyl)amino]propyloxypsoralen (14.0 mg, 57.8% yield),as a yellow solid. ¹H NMR (CDCl₃): d 1.97 (app quintet J=6.3 Hz, 2H),2.91 (app t, J=7.5 Hz, 6H), 3.55 (t, J=7.0 Hz, 4H), 4.58 (t, J=5.9 Hz,2H), 6.37 (d, J=9.5 Hz, 1H), 6.82 (d, J=2.1 Hz, 1H), 7.37 (s, 1H), 7.70(d, J=2.1 Hz, 1H), 7.77 (d, J=9.5 Hz, 1H).

[0233] 8-[5-(Bis-2-chloroethyl)amino]pentyloxypsoralen (compound 6)

[0234] In the same manner as the foregoing, but using 1,5-dibromopentanein Step 1 in place of 1,3-dibromopropane, tcompound 6 is produced.

Example 20

[0235] This example describes the synthesis of5-[3-(Bis-2-chloroethyl)aminopropyloxy]methyl-8-methoxypsoralen,(compound 4)

[0236] Step 1: 5-[(3-Hydroxy)-propyloxy]methyl-8-methoxypsoralen

[0237] 5-Bromomethyl-8-methoxypsoralen@ (0.300 g, 0.970 mmol) and1,3-propanediol (4 mL, 55.3 mmol) were refluxed in acetone (30 mL) for 3days. The solvent was removed under reduced pressure and the residue wasdissolved in CH₂Cl₂ and washed several times with water to remove excessdiol. After preliminary drying with brine then anhydrous Na₂SO₄, thesolvent was rotovapped off to give5-[(3-hydroxy)-propyloxy]methyl-8-methoxypsoralen (0.194 g, 65.8%yield), as a yellow solid. ¹H NMR (CDCl₃): d 1.86 (quintet, J=5.8 Hz,2H), 3.68 (t, J=5.9 Hz, 2H), 3.75 (t, J=5.8 Hz, 2H), 4.30 (s, 3H), 4.89(s, 2H), 6.43 (d, J=9.9 Hz, 1H), 6.95 (d, J=2.2 Hz, 1H), 7.71 (d, J=2.2Hz, 1H), 8.14 (d, J=9.9 Hz, 1H).

[0238] Step 2: 5-[(3-Methanesulfonyl)propyloxy]methyl-8-methoxypsoralen

[0239] A solution of 5-(1-hydroxy-propoxy)methyl-8-methoxy psoralen(0.194 g, 0.637 mmol) in CH₂Cl₂ (4 mL) was chilled with an ice/waterbath. Triethylamine (0.28 mL, 2.04 mmol) then methanesulfonyl chloride(0.15 mL,1.91 mmol) were added dropwise. The solution was allowed towarm to room temperature and stirred for 4-6 h. The reaction mixture waspartitioned between CH₂Cl₂ and water and the organic layer was washedseveral times with water, then brine, and dried with anhydrous Na₂SO₄and concentrated to give crude5-[(3-methanesulfonyl)propyloxy]methyl-8-methoxypsoralen (0.266 g, 109%yield) which was used directly for the next step.

[0240] Step 3:5-[3-(Bis-2-hydroxyethyl)aminopropyloxy]methyl-8-methoxypsoralen

[0241] A solution of5-[(3-methanesulfonyl)propyloxy]methyl-8-methoxypsoralen (0.266 g, 0.695mmol) and diethanolamine (0.500 g, 4.76 mmol) were refluxed inacetonitrile (6 mL) overnight. After rotovapping off the solvent, theresulting syrup was dissolved in CH₂Cl₂, washed with water severaltimes, then brine, dried with anhydrous Na₂SO₄ and stripped to give5-[3-(Bis-2-hydroxyethyl)aminopropyloxy]methyl-8-methoxypsoralen (0.217g, 82.8% yield), as a yellow solid. ¹H NMR (CDCl₃): d 1.76 (quintet,J=6.1 Hz, 2H), 2.60-2.67 (m, 6H), 3.55-3.63 (m, 6H), 4.29 (s, 3H), 4.88(s, 2H), 6.43 (d, J=9.9 Hz, 1H), 6.98 (d, J=2.1 Hz, 1H), 7.71 (d, J=2.2Hz, 1H), 8.15 (d, J=9.9 Hz, 1H).

[0242] Step 4:5-[3-(Bis-2-chloroethyl)aminopropyloxy]methyl-8-methoxypsoralen,(compound 4)

[0243] Thionyl chloride (0.040 mL, 0.55 mmol) was added dropwise to anice bath chilled solution of5-[3-(bis-2-hydroxyethyl)aminopropyloxy]methyl-8-methoxypsoralen (10.0mg, 0.0255 mmol) and pyridine (0.040 mL, 0.49 mmol) in CH₂Cl₂ (2 mL).The reaction mix was covered with a serum cap and allowed to stir 5 h atrt under nitrogen, then stripped under reduced vacuum and partitionedbetween CH₂Cl₂ and 10% NaHCO₃. The organic layer was washed severaltimes with aqueous NaHCO₃, then brine. After drying with anhydrousNa₂SO₄, solvent was rotovapped off to give5-[3-(bis-2-chloroethyl)aminopropyloxy]methyl-8-methoxypsoralen (0.0075mg, 63.0% yield), as a syrup. ¹H NMR (CDCl₃): d 1.71 (t, J=6.1 Hz, 2H),2.60 (t, J=6.4 Hz, 2H), 2.82 (t, J=6.6 Hz, 4H), 3.3-3.7 (m, 6H), 4.29(s, 3H), 4.86 (s, 2H), 6.43 (d, J=9.9 Hz, 1H), 6.96 (s, 1H), 7.71 (s,1H), 8.15 (d, J=9.9 Hz, 1H).

Example 21

[0244] This example describes the synthesis of5-[3-(Bis-2-chloroethyl)aminopropyloxy]methyl-8-methoxypsoralen,(compound 16)

[0245] Step 1:5′-[4-(Bis-2-hydroxyethyl)amino-1-butylaminomethyl]-4,4′,8-trimethylpsoralen

[0246] A solution of 5′-bromomethyl-4,4′,8-trimethylpsoralen (U.S. Pat.No. 4,294,822, 73.0 mg, 0.227 mmol), andN,N-bis(2-hydroxyethyl)-1,4-butanediamine (400 mg, 2.27 mmol) werestirred in acetonitrile (100 mL) for 4 h. After rotovapping off thesolvent, the resulting syrup was dissolved in CH₂Cl₂, washed with 0.3MHCl several times, then chilled in an ice/water bath and made basic withpowdered K₂CO₃. The product was extracted from the aqueous layer withseveral portions of CH₂Cl₂. The organic solution was rinsed with brinethen dried with anhydrous Na₂SO₄ and stripped to give a brown syrup. Thecrude product was chromatographed by TLC (silica gel, 1/9 MeOH/CHCl₃)dissolved in CH₂Cl₂ to give5′-[4-(bis-2-hydroxyethyl)amino-1-butylaminomethyl]-4,4′,8-trimethylpsoralen,as a yellow syrup (26.9 mg, 29% yield). ¹H NMR (CDCl₃): d 1.59 (s, 3H),2.49-2.76 (m, 14H), 3.62 (t, J=5.3 Hz, 4H), 3.97 (s, 2H), 6.23 (d, J=1.1Hz, 1H), 7.47 (s, 1H).

[0247] Step 2:5′-[4-(Bis-2-chloroethyl)amino-1-butylaminomethyl]-4,4′,8-trimethylpsoralen

[0248] Thionyl chloride (0.020 mL, 0.278 mmol) was added dropwise to anice bath chilled solution of5′-[3-(bis-2-hydroxyethyl)amino-1-butylaminomethyl]-4,4′,8-trimethylpsoralen(11.6 mg, 0.0278 mmol) and pyridine (0.023 mL, 0.278 mmol) in CH₂Cl₂ (5mL). The reaction mix was covered with a serum cap and allowed to stirovernight at rt, then stripped under reduced vacuum and partitionedbetween CH₂Cl₂ and 10% NaHCO₃. The organic layer was washed severaltimes with aqueous NaHCO₃, then brine. After drying with anhydrousNa₂SO₄, solvent was rotovapped off to give5′-[4-(bis-2-chloroethyl)amino-1-butylaminomethyl]-4,4′,8-trimethylpsoralen(9.3 mg, 73.8% yield), as a yellow solid. ¹H NMR (CDCl₃): d 1.54 (m,4H), 2.28 (s, 3H), 2.40-2.65 (m, 8H), 2.71 (t, J=6.8 Hz, 2H), 2.84 (t,J=7.0 Hz, 4H), 3.48 (t, J=7.0 Hz, 4 Hz), 3.97 (s, 2H) 6.24 (d, J=1.1 Hz,1H), 7.47 (s, 1H).

[0249]5′-[5-(Bis-2-chloroethyl)amino-1-pentylaminomethyl]-4,4′,8-trimethylpsoralen,(compound 17)

[0250] In the same manner as the foregoing, but usingN,N-bis(2-hydroxyethyl)-1,5-pentanediamine in Step 1 in place ofN,N-bis(2-hydroxyethyl)-1,4-butanediamine the title compound isproduced.

[0251]5′-[6-(Bis-2-chloroethyl)amino-1-hexylaminomethyl]-4,4′,8-trimethylpsoralen,(compound 18)

[0252] In the same manner as the foregoing, but usingN,N-bis(2-hydroxyethyl)-1,6-hexanediamine in Step 1 in place ofN,N-bis(2-hydroxyethyl)-1,4-butanediamine the title compound isproduced.

[0253]4′-[3-(Bis-2-chloroethyl)amino-1-propylaminomethyl]-4,5′,8-trimethylpsoralen,(compound 11)

[0254] In the same manner as the foregoing, but using4′-bromomethyl-4,5′,8-trimethylpsoralen (U.S. Pat. No. 4,124,598) andN,N-bis(2-hydroxyethyl)-1,3-propanediamine in Step 1 in place of5′-bromomethyl-4,4′,8-trimethylpsoralen andN,N-bis(2-hydroxyethyl)-1,4-butanediamine respectively, the titlecompound is produced.

[0255]4′-[4-(Bis-2-chloroethyl)amino-1-butylaminomethyl]-4,5′,8-trimethylpsoralen,(compound 12)

[0256] In the same manner as the foregoing, but using4′-bromomethyl-4,5′,8-trimethylpsoralen andN,N-bis(2-hydroxyethyl)-1,4-butanediamine in Step 1 in place of5′-bromomethyl-4,4′,8-trimethylpsoralen, the title compound is produced.

[0257]4′-[3-(Bis-2-chloroethyl)amino-1-hexylaminomethyl]-4,5′,8-trimethylpsoralen,(compound 13)

[0258] In the same manner as the foregoing, but using4′-bromomethyl-4,5′,8-trimethylpsoralen andN,N-bis(2-hydroxyethyl)-1,6-hexanediamine in Step 1 in place of5′-bromomethyl-4,4′,8-trimethylpsoralen andN,N-bis(2-hydroxyethyl)-1,4-butanediamine respectively, the titlecompound is produced.

Example 22

[0259] This example describes the synthesis of4′-[4-(Bis-2-chloroethyl)aminobutoxy]methyl-4,5′,8-trimethylpsoralen,(compound 9)

[0260] Step 1:4′-[(4-Methanesulfonyl)-butoxy]methyl-4,5′,8-trimethylpsoralen

[0261] A solution of4′-[(4-hydroxy)-butoxy]methyl-4,5′,8-trimethylpsoralen (1-U.S. Pat. No.4,269,852, 91.5 mg, 0.301 mmol) in CH₂Cl₂ (5 mL) was chilled with anice/water bath. Triethylamine (0.14 mL, 1.00 mmol) then methanesulfonylchloride (0.070 mL, 0.903 mmol) were added dropwise. The solution wasallowed to warm to room temperature and stirred overnight. The reactionmixture was partitioned between CH₂Cl₂ and water. The organic layer waswashed several times with aqueous NaHCO₃, then brine, and dried withanhydrous Na₂SO₄ and concentrated to give crude4′-[(4-methanesulfonyl)-butoxy]methyl-4,5′,8-trimethylpsoralen (0.106 g,86.2% crude yield). ¹H NMR (CDCl₃): d 1.71-1.86 (m, 4H), 2.47 (s, 6H),2.52 (s, 3H), 2.95 (s, 3H), 3.51 (t, J=6.0, 2H), 4.21 (t, J=6.2, 2H),4.59 (s, 2H), 6.20 (s, 1H), 7.54 (s, 1H).

[0262] Step 2:4′-[4-(Bis-2-hydroxyethyl)aminobutoxy]methyl-4,5′,8-trimethylpsoralen

[0263] A solution of crude4′-[(4-methanesulfonyl)-butoxy]methyl-4,5′,8-trimethylpsoralen (106 mg,0.260 mmol) and diethanolamine (300 mg, 2.85 mmol) were refluxed inacetonitrile (8 mL) overnight. After rotovapping off the solvent, theresulting syrup was dissolved in CH₂Cl₂, washed several times withaqueous NaHCO₃, then brine, and dried with anhydrous Na₂SO₄ andconcentrated to give crude product which was chromatographed by TLC(silica gel, 95/5 CHCl₃—MeOH) to give a4′-[4-(bis-2-hydroxyethyl)aminobutoxy]methyl-4,5′,8-trimethylpsoralen asa yellow solid (32 mg, 27.1% yield). ¹H NMR (CDCl₃): d 1.49-1.75 (m,4H), 2.40-2.70 (m, 15H), 3.48 (t, J=5.8, 2H), 3.59 (t, J=5.3, 2H), 4.61)s, 2H), 6.24 (d, J=1.1 Hz, 1H), 7.60 (s, 1H).

[0264] Step 3:4′-[4-(Bis-2-chloroethyl)aminobutoxy]methyl-4,5′,8-trimethylpsoralen,(compound 9)

[0265] Thionyl chloride (0.040 mL, 0.55 mmol) was added dropwise to anice bath chilled solution of4′-[4-(bis-2-hydroxyethyl)aminobutyloxy]methyl-4,5′,8-trimethylpsoralen(24.0 mg, 0.0575 mmol) and pyridine (0.050 mL, 0.62 mmol) in CH₂Cl₂ (3mL). The reaction mix was covered with a serum cap and allowed to stirovernight at rt, then stripped under reduced vacuum and partitionedbetween CH₂Cl₂ and 10% NaHCO₃. The organic layer was washed severaltimes with aqueous NaHCO₃, then brine. After drying with anhydrousNa₂SO₄, solvent was rotovapped off to give4′-[4-(bis-2-chloroethyl)aminobutoxy]methyl-4,5′,8-trimethylpsoralen(20.5 mg, 78.5% yield), as a yellow solid. ¹H NMR (CDCl₃): d 1.40-1.74(m, 4H), 2.45-2.65 (m, 11H), 2.81 (t, J=7.0 Hz, 4H), 3.37-3.52 (m, 6H),4.61 (s, 2H), 6.24 (s,1H), 7.59 (s, 1H).

[0266] It is to be understood that the present invention is not to belimited to the exact details of operation or exact compounds,compositions, methods, or procedures shown and described, asmodifications and equivalents will be apparent to one skilled in theart. All patents described are hereby incorporated by reference. TABLE24 EXAMPLE 23 Dose for Logs Virus Killed at Fixed Drug Dose Compound 5log kill CA M = N(CH₂CH₂Cl)₂ (μM) R-17 R-17 R-17 HIV NA = not testedR-17 0.5 μM 2 μM 5 μM 15 μM QM ≦0.5 >6 >6 >6 2.8-4.2

>30 <0.5 <0.5 0.5 NA

>75 <0.5 <0.5 <0.5 NA

12-25 <0.5 0.5 2-3 NA

10 <0.5 <0.5 2-3 NA

>30 <0.5 <0.5 1 NA

[0267] TABLE 25 Dose for Logs Virus Killed at Fixed Drug Dose Compound 5log kill CA- M = N(CH₂CH₂Cl)₂ (μM) R-17 R-17 R-17 HIV NA = not testedR-17 0.5 μM 2 μM 5 μM 15 μM

2 2 5 >6 0

7 ? 3 4 0.2

NA NA NA NA NA

[0268] TABLE 26 Dose for Logs Virus Killed at Fixed Drug Dose Compound 5log kill CA- M = N(CH₂CH₂Cl)₂ (μM) R-17 R-17 R-17 HIV NA = not testedR-17 0.5 μM 2 μM 5 μM 15 μM

>30 0.5 NA

≦2 1 6 >6 0.4

0.8

≦2 ? >6 >6 1.5

1.0

0.3

[0269] TABLE 27 Dose for Logs Virus Killed at Fixed Drug Dose 5 log killCA (μM) R-17 R-17 R-17 HIV Compound ^((a)) R-17 0.5 μM 2 μM 5 μM 15 μM

≦2 ? >6 >6 0.7 n = 5 compound 17 n = 6 compound 18

We claim:
 1. A method of inactivating a pathogen in mammalian blood or amammalian blood product comprising: (a) contacting, in vitro, mammalianblood or a mammalian blood product with a pathogen inactivating amountof a first compound that binds nucleic acid non-covalently and has amoiety selected from the group consisting of a mustard group, anaziridinium group, and an aziridine group, wherein the first compoundhas a greater inactivation efficiency against R17 than a second compoundhaving the mustard group, aziridinium group, or aziridine group thatdoes not bind nucleic acid non-covalently, and wherein the pathogeninactivating amount results in the inactivation of at least 1 log of apathogen present in the blood or blood product, if any; and (b)recovering the pathogen inactivated blood or blood product, wherein therecovered blood or blood product is suitable for therapeutic use in amammal.
 2. The method according to claim 1, wherein the first compoundbinds nucleic acid non-covalently by a mode selected from the groupconsisting of intercalating, minor groove binding, major groove binding,phosphate backbone binding, and sequence specific binding.
 3. The methodaccording to claim 2 wherein the pathogen is an RNA containing pathogen.4. The method according to claim 3 wherein the pathogen is HIV.
 5. Themethod according to claim 2 wherein the pathogen is a DNA containingpathogen.
 6. The method according to claim 5 wherein the pathogen is ahepatitis virus.
 7. The method according to claim 2, wherein the firstcompound comprises a polyamine.
 8. The method according to claim 7,wherein the moiety is an aziridine.
 9. The method according to claim 8,wherein the blood product comprises red blood cells.
 10. The methodaccording to claim 8, wherein the blood product is whole blood.